Methods For Degrading or Converting Plant Cell Wall Polysaccharides

ABSTRACT

The present invention relates to methods for converting plant cell wall polysaccharides into one or more products, comprising: treating the plant cell wall polysaccharides with an effective amount of a spent whole fermentation broth of a recombinant microorganism, wherein the recombinant microorganism expresses one or more heterologous genes encoding enzymes which degrade or convert the plant cell wall polysaccharides into the one or more products. The present invention also relates to methods for producing an organic substance, comprising: (a) saccharifying plant cell wall polysaccharides with an effective amount of a spent whole fermentation broth of a recombinant microorganism, wherein the recombinant microorganism expresses one or more heterologous genes encoding enzymes which degrade or convert the plant cell wall polysaccharides into saccharified material; (b) fermenting the saccharified material of step (a) with one or more fermenting microoganisms; and (c) recovering the organic substance from the fermentation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/172,852, filed Jul. 14, 2008, which is a divisional of U.S.application Ser. No. 11/078,921, filed Mar. 10, 2005, now U.S. Pat. No.7,413,882, which claims the benefit of U.S. Provisional Application No.60/556,779, filed Mar. 25, 2004, which applications are incorporatedherein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under NREL SubcontractNo. ZCO-30017-02, Prime Contract DE-AC36-98GO10337 awarded by theDepartment of Energy. The government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for degrading or convertingplant cell wall polysaccharides and to products obtained by suchmethods.

2. Description of the Related Art

Plant cell walls are composed of a mixture of polysaccharidesinterlocked in a complex structure (Carpita et al., 2001, PlantPhysiology 127: 551-565). The mixture of polysaccharides includecellulose, xyloglycan (hemicellulose), and pectic polymers, which areprimarily composed of hexoses, e.g., glucose, galactose, and mannose;pentoses, e.g., xylose and arabinose; uronic acids, e.g., galacturonicacid and glucuronic acid; and deoxyhexoses, e.g., rhamnose and fucose.

Plant cell wall polysaccharides can be enzymatically degraded toglucose, xylose, mannose, galactose, and arabinose, which can then beconverted to other organic substances, for example, glucose is easilyfermented by yeast into ethanol. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes can be used as sources of plant cellwall polysaccharides.

Cellulose is a primary component of plant cell walls. Manymicroorganisms produce enzymes that degrade cellulose. These enzymesinclude, for example, endoglucanases, cellobiohydrolases, andbeta-glucosidases. Endoglucanases digest the cellulose polymer at randomlocations, opening it to attack by cellobiohydrolases.Cellobiohydrolases sequentially release molecules of cellobiose from theends of the cellulose polymer. Cellobiose is a water-solublebeta-1,4-linked dimer of glucose. Beta-glucosidases hydrolyze cellobioseto glucose.

Natural microorganisms that degrade cellulose and other cell wallpolysaccharides may not be ideal for large-scale conversion ofcellulosic materials because (a) the full complement of enzymes may belacking, (b) one or more enzyme components perform poorly, are labile,or their kinetic behavior fails to meet the specification of theintended use, (c) the conversion and/or degradation could be improved byexpression of a heterologous enzyme gene that enhances theconversion/degradation, or (d) the full complement of enzymes may be ininsufficient amounts to be economically viable. It would be an advantageto the art to improve the degradation and conversion of plant cell wallpolysaccharides by using whole fermentation broth from recombinantmicroorganisms to circumvent expensive cell removal and enzymeformulation steps.

It is an object of the present invention to provide new methods fordegrading or converting plant cell wall polysaccharides into variousproducts using spent whole fermentation broths from recombinantmicroorganisms.

SUMMARY OF THE INVENTION

The present invention relates to methods for degrading or convertingplant cell wall polysaccharides into one or more products, comprising:treating the plant cell wall polysaccharides with an effective amount ofa spent whole fermentation broth of a recombinant microorganism, whereinthe recombinant microorganism expresses one or more heterologous genesencoding enzymes which degrade or convert the plant cell wallpolysaccharides into the one or more products.

The present invention also relates to methods for producing one or moreorganic substances, comprising:

(a) saccharifying plant cell wall polysaccharides with an effectiveamount of a spent whole fermentation broth of a recombinantmicroorganism, wherein the recombinant microorganism expresses one ormore heterologous genes encoding enzymes which degrade or convert theplant cell wall polysaccharides into saccharified material;

(b) fermenting the saccharified material of step (a) with one or morefermenting microoganisms; and

(c) recovering the one or more organic substances from the fermentation.

The present invention further relates to products or organic substancesobtained by such methods. In a preferred aspect, the organic substanceis alcohol.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction map of pAlLo01.

FIG. 2 shows a restriction map of pMJ04.

FIG. 3 shows a restriction map of pCaHj527.

FIG. 4 shows a restriction map of pMT2188.

FIG. 5 shows a restriction map of pCaHj568.

FIG. 6 shows a restriction map of pMJ05.

FIG. 7 shows a restriction map of pSMai130.

FIG. 8 shows the DNA sequence (SEQ ID NO: 32) and deduced amino acidsequence (SEQ ID NO: 33) of the secretion signal sequence of anAspergillus oryzae beta-glucosidase.

FIG. 9 shows the DNA sequence (SEQ ID NO: 36) and deduced amino acidsequence (SEQ ID NO: 37) of the secretion signal sequence of a Humicolainsolens endoglucanase V.

FIG. 10 shows a restriction map of pSMai135.

FIG. 11 shows the PCS hydrolysis profiles of whole fermentation broth(WB) (panel A) and cell-free broth (CB) (panel B) at enzyme dosesranging from 2.5 to 20 mg/g of PCS (noted in the lower right of eachpanel).

FIG. 12 shows the PCS hydrolysis curves for WB and CB samples derivedfrom freshly harvested Trichoderma reesei RutC30 fermentation material.Each profile is plotted as % RS yield (% of theoretical maximum reducingsugar based on the glucan composition of 10 mg of PCS per ml) as afunction of hydrolysis time (1-120 hours). Enzyme doses are noted in theupper right of each panel.

FIG. 13 shows a comparison of total reducing sugar (RS) and glucoseliberated during PCS hydrolysis reactions using WB and CB samples fromTrichoderma reesei RutC30. Enzyme doses are noted at the top of eachpanel. The sample numbers noted on the X-axis correspond to hydrolysistimes spanning 1 to 120 hours.

FIG. 14 shows the PCS hydrolysis curves for WB and CB samples derivedfrom freshly harvested Trichoderma reesei SMA135-04 fermentation broth.Trichoderma reesei strain SMA135-04 expresses recombinant Aspergillusoryzae beta-glucosidase. Each profile is plotted as % RS yield (% oftheoretical maximum reducing sugar based on the glucan composition of 10mg of PCS per ml) as a function of hydrolysis time (1-120 hours). Enzymedoses are noted in the upper right of each panel.

FIG. 15 shows a comparison of total reducing sugar (RS) and glucoseliberated during PCS hydrolysis reactions using WB and CB samples fromTrichoderma reesei SMA135-04 that harbors an expression vector directingsynthesis and secretion of Aspergillus oryzae beta-glucosidase. Enzymedoses are noted at the top of each panel. The sample numbers noted onthe X-axis correspond to hydrolysis times spanning 1 to 120 hours.

FIG. 16 shows the PCS hydrolysis curves for WB and CB samples derivedfrom Trichoderma reesei RutC30 fermentation broth stored two weeks at 4°C. Each profile is plotted as % RS yield (% of theoretical maximumreducing sugar based on the glucan composition of 10 mg of PCS per ml)as a function of hydrolysis time (1-120 hours). Enzyme doses are notedin the upper right of each panel.

FIG. 17 shows the PCS hydrolysis curves for WB and CB samples derivedfrom Trichoderma reesei SMA135-04 fermentation broth stored two weeks at4° C. Each profile is plotted as % RS yield (% of theoretical maximumbased on the glucan composition of 10 mg/ml PCS) as a function ofhydrolysis time (1-120 hours). Enzyme doses are noted in the upper rightof each panel.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for degrading or convertingplant cell wall polysaccharides into one or more products, comprising:treating the plant cell wall polysaccharides with an effective amount ofa spent whole fermentation broth of a recombinant microorganism, whereinthe recombinant microorganism expresses one or more heterologous genesencoding enzymes which degrade or convert the plant cell wallpolysaccharides into the one or more products. The present inventionalso relates to methods for producing one or more organic substances,comprising: (a) saccharifying plant cell wall polysaccharides with aneffective amount of a spent whole fermentation broth of a recombinantmicroorganism, wherein the recombinant microorganism expresses one ormore heterologous genes encoding enzymes which degrade or convert theplant cell wall polysaccharides into one or more products; (b)fermenting the saccharified material of step (a) with one or morefermenting microoganisms; and (c) recovering the one or more organicsubstances from the fermentation.

Plant Cell Wall Polysaccharides

In the methods of the present invention, the source of the plant cellwall polysaccharides can be any plant biomass containing cell wallpolysaccharides. Such sources include, but are not limited to,herbaceous material, agricultural residues, forestry residues, municipalsolid waste, waste paper, and pulp and paper mill residues.

In a preferred aspect, the plant cell wall biomass is corn stover. Inanother preferred aspect, the plant cell wall biomass is corn fiber. Inanother preferred aspect, the plant cell wall biomass is rice straw. Inanother preferred aspect, the plant cell wall biomass is paper and pulpprocessing waste. In another preferred aspect, the plant cell wallbiomass is woody or herbaceous plants. In another preferred aspect, theplant cell wall biomass is fruit pulp. In another preferred aspect, theplant cell wall biomass is vegetable pulp. In another preferred aspect,the plant cell wall biomass is pumice. In another preferred aspect, theplant cell wall biomass is distillers grain.

The plant cell wall biomass may be used as is or may be subjected topretreatment using conventional methods known in the art. Suchpretreatments includes physical, chemical, and biological pretreatment.For example, physical pretreatment techniques can include various typesof milling, crushing, irradiation, steaming/steam explosion, andhydrothermolysis. Chemical pretreatment techniques can include diluteacid, alkaline, organic solvent, ammonia, sulfur dioxide, carbondioxide, and pH-controlled hydrothermolysis. Biological pretreatmenttechniques can involve applying lignin-solubilizing microorganisms (see,for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook onBioethanol: Production and Utilization, Wyman, C. E., ed., Taylor &Francis, Washington, D.C., 179-212; Ghosh, P., Singh, A., 1993,Physicochemical and biological treatments for enzymatic/microbialconversion of lignocellulosic biomass, Adv. Appl. Microbiol., 39:295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass: areview, in Enzymatic Conversion of Biomass for Fuels Production, Himmel,M. E., Baker, J. O., and Overend, R. P., eds., ACS Symposium Series 566,American Chemical Society, Washington, D.C., chapter 15; Gong, C. S.,Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production fromrenewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Olsson, L., and Hahn-Hagerdal, B.,1996, Fermentation of lignocellulosic hydrolysates for ethanolproduction, Enz. Microb. Tech, 18: 312-331; and Vallander, L., andEriksson, K.-E. L., 1990, Production of ethanol from lignocellulosicmaterials: State of the art, Adv. Biochem. Eng./Biotechnol., 42: 63-95).

In the present invention, the plant cell wall polysaccharides include,but are not limited to, cellulose, hemicellulose, and pectic substances.

Cellulose is composed of beta-1,4-glucan. Hemicellulose is composed ofbeta-1,3-1,4-glucan, xyloglucan, xylan (arabinoxylan), mannan(galactomannan), galactan (arabinogalactan), and arabinan. Pecticsubstances are composed of homogalacturonan (pectin),rhamnogalacturonan, and xylogalacturonan.

Beta-1,4-glucan is composed of beta-1,4-linked glucose. Enzymes thatdegrade beta-1,4-glucan include endoglucanase, cellobiohydrolase, andbeta-glucosidase.

Beta-1,3-1,4-glucan is composed of beta-1,4-linked glucose interruptedby beta-1,3-linked glucose. Enzymes that degrade beta-1,3-1,4-glucaninclude endo-beta-1,3(4)-glucanase, endoglucanase (beta-glucanase,cellulase), and beta-glucosidase.

Xyloglucans are composed of beta-1,4-linked glucose withalpha-1,6-linked xylose substituents. Enzymes that degrade xyloglucansinclude xyloglucanase, endoglucanase, and cellulase.

Xylan (arabinoxylan) is composed of beta-1,4-linked xylose, withalpha-1,2 or alpha-1,3 linked arabinoses. The xylose can be acetylated.Glucuronic acid is also present. Enzymes that degrade xylan includexylanase, xylosidase, alpha-arabinofuranosidase, alpha-glucuronidase,and acetyl xylan esterase.

Mannan (galactomannan) is composed of beta-1,4-linked mannose withalpha-1,6-linked galactose substituents. The mannose substituents canalso be acetylated. Enzymes that degrade mannan include mannanase,mannosidase, alpha-galactosidase, and mannan acetyl esterase.

Galactan (arabinogalactan) is composed of D-galactose and3,6-anhydrogalactose linked by beta-1,3-linkages. Enzymes that degradegalactan include galactanases.

Arabinan is composed of 1,3-1,5-linked L-arabinose. Enzymes that degradearabinan include arabinanases.

Homogalacturonan is composed of alpha-1,4-linked galacturonic acid. Thegalacturonic acid substituents may be acetylated and/or methylated.Enzymes that degrade homogalacturonan include pectate lyase, pectinlyase, pectate lyase, polygalacturonase, pectin acetyl esterase, andpectin methyl esterase.

Rhamnogalacturonan is composed of alternating alpha-1,4-rhamnose andalpha-1,2-linked galacturonic acid, with side chains linked 1,4 torhamnose. The side chains include Type I galactan, which isbeta-1,4-linked galactose with alpha-1,3-linked arabinose substituents;Type II galactan, which is beta-1,3-1,6-linked galactoses (verybranched) with arabinose substituents; and arabinan, which isalpha-1,5-linked arabinose with alpha-1,3-linked arabinose branches. Thegalacturonic acid substituents may be acetylated and/or methylated.Enzymes that degrade rhamnogalacturonan includealpha-arabinofuranosidase, beta-galactosidase, galactanase, arabinanase,alpha-arabinofuranosidase, rhamnogalacturonase, rhamnogalacturonanlyase, and rhamnogalacturonan acetyl esterase.

Xylogalacturonan is composed of alpha-1,4-linked galacturonic acid withside chains of xylose. Galactose and fucose may be linked to the xylosesubstituents. Rhamnose is also present. The galacturonic acidsubstituents may be acetylated and/or methylated. Enzymes that degradexylogalacturonan include xylogalacturonosidase, xylogalacturonase, andrhamnogalacturonan lyase.

Cellulose may also be present as lignocellulose. Lignin is composed ofmethoxylated phenyl-propane units linked by ether linkages and C—Cbonds. The chemical composition of lignin differs according to the plantspecies. Such components include guaiacyl, 4-hydroxyphenyl, and syringylgroups. Enzymes that degrade the lignin component of lignocelluloseinclude lignin peroxidases, manganese-dependent peroxidases, hybridperoxidases, with combined properties of lignin peroxidases andmanganese-dependent peroxidases, and laccases (Vicuna, 2000, MolecularBiotechnology 14: 173-176; Broda et al., 1996, Molecular Microbiology19: 923-932).

Recombinant Microorganisms

In the methods of the present invention, the recombinant microorganismcan be any microorganism that is useful as a host for the recombinantproduction of enzymes useful in the conversion or degradation of plantcell wall polysaccharides. The microorganism chosen as a host forrecombinant production may already contain one or more native genesencoding enzymes that degrade or convert plant cell wallpolysaccharides. However, the host may be deficient in the fullcomplement of enzymes necessary to degrade or convert plant cell wallpolysaccharides, i.e., the host may lack one or more genes.Alternatively, the host may contain the full complement of enzymes, butone or more enzymes may be poorly expressed. Moreover, the host may lackone or more genes required to produce the full complement of enzymes andone or more enzymes the host does produce may be poorly expressed. Itwill be understood in the present invention that a gene native to thehost that has undergone manipulation, as described herein, will beconsidered a heterologous gene.

The host is preferably a fungal strain. “Fungi” as used herein includesthe phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (asdefined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary ofThe Fungi, 8th edition, 1995, CAB International, University Press,Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al.,1995, supra, page 171) and all mitosporic fungi (Hawksworth et al.,1995, supra).

In a preferred aspect, the fungal host is a yeast strain. “Yeast” asused herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In a more preferred aspect, the yeast host is a Candida, Hansenula,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiastrain.

In a most preferred aspect, the yeast host is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis strain. In another most preferredaspect, the yeast host is a Kluyveromyces lactis strain. In another mostpreferred aspect, the yeast host is a Yarrowia lipolytica strain.

In another preferred aspect, the fungal host is a filamentous fungalstrain. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In a more preferred aspect, the filamentous fungal host is, but notlimited to, an Acremonium, Aspergillus, Fusarium, Humicola, Mucor,Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia,Tolypocladium, or Trichoderma strain.

In an even more preferred aspect, the filamentous fungal host is anAspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae strain.In another even more preferred aspect, the filamentous fungal host is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum strain. In another evenmore preferred aspect, the filamentous fungal host is a Humicolainsolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Scytalidium thermophilum,or Thielavia terrestris strain. In a further even more preferred aspect,the filamentous fungal host is a Trichoderma harzianum, Trichodermakoningii, Trichoderma longibrachiatum, Trichoderma reesei, orTrichoderma viride strain.

In a most preferred aspect, the filamentous fungal host is Trichodermareesei RutC30, which is available from the American Type CultureCollection as Trichoderma reesei ATCC 56765.

In a preferred aspect, the host or recombinant microorganism comprisesone or more heterologous genes encoding enzymes selected from the groupconsisting of endoglucanase (cellulase), cellobiohydrolase, andbeta-glucosidase.

In a more preferred aspect, the recombinant microorganism comprises aheterologous gene encoding an endoglucanase. In another more preferredaspect, the recombinant microorganism comprises a heterologous geneencoding a cellobiohydrolase gene. In another more preferred aspect, therecombinant microorganism comprises a heterologous gene encoding abeta-glucosidase.

In a most preferred aspect, the recombinant microorganism comprisesheterologous genes encoding an endoglucanase and a cellobiohydrolase. Inanother most preferred aspect, the recombinant microorganism comprisesheterologous genes encoding an endoglucanase and a beta-glucosidase.

In another most preferred aspect, the recombinant microorganismcomprises heterologous genes encoding an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

In another preferred aspect, the recombinant microorganism furthercomprises a glucohydrolase.

In another preferred aspect, the recombinant microorganism furthercomprises one or more heterologous genes encoding enzymes selected fromthe group consisting of xyloglucanase, xylanase, xylosidase,alpha-arabinofuranosidase, alpha-glucuronidase, and acetyl xylanesterase.

In another preferred aspect, the recombinant microorganism furthercomprises one or more heterologous genes encoding enzymes selected fromthe group consisting of mannanase, mannosidase, alpha-galactosidase,mannan acetyl esterase, galactanase, and arabinanase.

In another preferred aspect, the recombinant microorganism furthercomprises one or more heterologous genes encoding enzymes selected fromthe group consisting of pectate lyase, pectin lyase, polygalacturonase,pectin acetyl esterase, pectin methyl esterase,alpha-arabinofuranosidase, beta-galactosidase, galactanase, arabinanase,alpha-arabinofuranosidase, rhamnogalacturonase, rhamnogalacturonanlyase, rhamnogalacturonan acetyl esterase, xylogalacturonosidase,xylogalacturonase, and rhamnogalacturonan lyase.

In another preferred aspect, the recombinant microorganism furthercomprises one or more heterologous genes encoding enzymes selected fromthe group consisting of a lignin peroxidase, manganese-dependentperoxidase, and hybrid peroxidase.

In another preferred aspect, the recombinant microorganism even furthercomprises one or more heterologous genes encoding enzymes selected fromthe group consisting of an esterase, lipase, oxidase, phospholipase,phytase, protease, and peroxidase.

A gene encoding a plant cell wall degrading or converting enzyme may beof fungal or bacterial origin, e.g., species of Humicola, Coprinus,Thielavia, Fusarium, Myceliophthora, Acremonium, Cephalosporium,Scytalidium, Penicillium or Aspergillus (see, for example, EP 458162),especially those selected from the species Humicola insolens(reclassified as Scytalidium thermophilum, see for example, U.S. Pat.No. 4,435,307), Coprinus cinereus, Fusarium oxysporum, Myceliophthorathermophila, Meripilus giganteus, Thielavia terrestris, Acremonium sp.,Acremonium persicinum, Acremonium acremonium, Acremonium brachypenium,Acremonium dichromosporum, Acremonium obclavatum, Acremoniumpinkertoniae, Acremonium roseogriseum, Acremonium incoloratum, andAcremonium furatum; preferably from the species Humicola insolens DSM1800, Fusarium oxysporum DSM 2672, Myceliophthora thermophila CBS117.65, Cephalosporium sp. RYM-202, Acremonium sp. CBS 478.94,Acremonium sp. CBS 265.95, Acremonium persicinum CBS 169.65, Acremoniumacremonium AHU 9519, Cephalosporium sp. CBS 535.71, Acremoniumbrachypenium CBS 866.73, Acremonium dichromosporum CBS 683.73,Acremonium obclavatum CBS 311.74, Acremonium pinkertoniae CBS 157.70,Acremonium roseogriseum CBS 134.56, Acremonium incoloratum CBS 146.62,and Acremonium furatum CBS 299.70H. Plant cell wall hydrolytic enzymegenes may also be obtained from Trichoderma (particularly Trichodermaviride, Trichoderma reesei, and Trichoderma koningii), alkalophilicBacillus (see, for example, U.S. Pat. No. 3,844,890 and EP 458162), andStreptomyces (see, for example, EP 458162).

The enzymes and genes thereof referenced herein may be obtained from anysuitable origin, including, bacterial, fungal, yeast or mammalianorigin. The term “obtained” as used herein in connection with a givensource shall mean that the polypeptide encoded by a nucleotide sequenceis produced by the source or by a strain in which the nucleotidesequence from the source has been inserted. Encompassed within themeaning of a native enzyme are natural variants or variants obtained,for example, by site-directed mutagenesis or shuffling.

Techniques used to isolate or clone a gene encoding an enzyme are knownin the art and include isolation from genomic DNA, preparation fromcDNA, or a combination thereof. The cloning of a gene from such genomicDNA can be effected, e.g., by using the well known polymerase chainreaction (PCR) or antibody screening of expression libraries to detectcloned DNA fragments with shared structural features. See, e.g., Inniset al., 1990, PCR: A Guide to Methods and Application, Academic Press,New York. Other nucleic acid amplification procedures such as ligasechain reaction (LCR), ligated activated transcription (LAT) andnucleotide sequence-based amplification (NASBA) may be used.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host strains are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Enzymes Having Plant Cell Wall Hydrolytic Activity and Genes Thereof

In the methods of the present invention, the recombinant microorganismcomprises one or more genes which are heterologous or foreign to themicroorganism, wherein the one or more genes encode enzymes involved inthe degradation or conversion of plant cell wall polysaccharides.

The heterologous genes may encode enzymes that degrade beta-1,4-glucansuch as endoglucanase (cellulase), cellobiohydrolase, glucohydrolase,and beta-glucosidase; degrade beta-1,3-1,4-glucan such asendo-beta-1,3(4)-glucanase, endoglucanase (beta-glucanase, cellulase),and beta-glucosidase; degrade xyloglucans such as xyloglucanase,endoglucanase, and cellulase; degrade xylan such as xylanase,xylosidase, alpha-arabinofuranosidase, alpha-glucuronidase, and acetylxylan esterase; degrade mannan such as mannanase, mannosidase,alpha-galactosidase, and mannan acetyl esterase; degrade galactan suchas galactanase; degrade arabinan such as arabinanase; degradehomogalacturonan such as pectate lyase, pectin lyase, pectate lyase,polygalacturonase, pectin acetyl esterase, and pectin methyl esterase;degrade rhamnogalacturonan such as alpha-arabinofuranosidase,beta-galactosidase, galactanase, arabinanase, alpha-arabinofuranosidase,rhamnogalacturonase, rhamnogalacturonan lyase, and rhamnogalacturonanacetyl esterase; degrade xylogalacturonan such as xylogalacturonosidase,xylogalacturonase, and rhamnogalacturonan lyase; and degrade lignin suchas lignin peroxidases, manganese-dependent peroxidases, hybridperoxidases, with combined properties of lignin peroxidases andmanganese-dependent peroxidases, and laccases.

Genes encoding polysaccharide-degrading enzymes may be obtained fromsources as described by B. Henrissat, 1991, A classification of glycosylhydrolases based on amino-acid sequence similarities, Biochem. J. 280:309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696., which is incorporated herein by reference.

The recombinant microorganism may further comprise one or moreheterologous genes encoding enzymes such as esterases, lipases,oxidases, phospholipases, phytases, proteases, and peroxidases.

The enzymes may have activity either in the acid, neutral, or alkalinepH-range. In a preferred aspect, the enzymes have activity in the pHrange of about 2 to about 7.

Endoglucanases

The term “endoglucanase” is defined herein as anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. No. 3.2.1.4)which catalyses endohydrolysis of 1,4-beta-D-glycosidic linkages incellulose, cellulose derivatives (such as carboxymethyl cellulose andhydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3glucans such as cereal beta-D-glucans or xyloglucans, and other plantmaterial containing cellulosic components. For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) hydrolysis according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268.

In a preferred aspect, an endoglucanase gene is obtained from aTrichoderma reesei strain. In another preferred aspect, an endoglucanasegene is obtained from an Aspergillus oryzae strain. In another preferredaspect, an endoglucanase gene is obtained from an Aspergillus aculeatusstrain. In another preferred aspect, an endoglucanase gene is obtainedfrom a Humicola insolens strain.

Preferred examples of endoglucanase genes that can be used in theinvention are obtained from Aspergillus aculeatus (U.S. Pat. No.6,623,949; WO 94/14953), Aspergillus kawachii (U.S. Pat. No. 6,623,949),Aspergillus oryzae (Kitamoto et al., 1996, Appl. Microbiol. Biotechnol.46: 538-544; U.S. Pat. No. 6,635,465), Aspergillus nidulans (Lockingtonet al., 2002, Fungal Genet. Biol. 37: 190-196), Cellulomonas fimi (Wonget al., 1986, Gene 44: 315-324), Bacillus subtilis (MacKay et al., 1986,Nucleic Acids Res. 14: 9159-9170), Cellulomonas pachnodae (Cazemier etal., 1999, Appl. Microbiol. Biotechnol. 52: 232-239), Fusarium equiseti(Goedegebuur et al., 2002, Curr. Genet. 41: 89-98), Fusarium oxysporum(Hagen et al., 1994, Gene 150: 163-167; Sheppard et al., 1994, Gene 150:163-167), Humicola insolens (U.S. Pat. No. 5,912,157; Davies et al.,2000, Biochem J. 348: 201-207), Hypocrea jecorina (Penttila et al.,1986, Gene 45: 253-263), Humicola grisea (Goedegebuur et al., 2002,Curr. Genet. 41: 89-98), Micromonospora cellulolyticum (Lin et al.,1994, J. Ind. Microbiol. 13: 344-350), Myceliophthora thermophila (U.S.Pat. No. 5,912,157), Rhizopus oryzae (Moriya et al., 2003, J. Bacteriol.185: 1749-1756), Trichoderma reesei (Saloheimo et al., 1994, Mol.Microbiol. 13: 219-228), and Trichoderma viride (Kwon et al., 1999,Biosci. Biotechnol. Biochem. 63: 1714-1720; Goedegebuur et al., 2002,Curr. Genet. 41: 89-98).

Cellobiohydrolases

Cellobiohydrolase, an exo-1,4-beta-D-glucan cellobiohydrolase (E.C.3.2.1.91), catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing ornon-reducing ends of the chain. For purposes of the present invention,cellobiohydrolase activity is determined according to the proceduresdescribed by Lever et al., 1972, Anal. Biochem. 47: 273-279; vanTilbeurgh et al., 1982, FEBS Letters, 149: 152-156; and van Tilbeurghand Claeyssens, 1985, FEBS Letters, 187: 283-288. In the presentinvention, the Lever et al. method is employed to assess hydrolysis ofcellulose in corn stover, while the methods of van Tilbeurgh et al. areused to determine the cellobiohydrolase activity on a fluorescentdisaccharide derivative.

In a preferred aspect, a cellobiohydrolase gene is obtained from aTrichoderma reesei strain. In another preferred aspect, acellobiohydrolase gene is obtained from an Aspergillus aculeatus strain.In another preferred aspect, a cellobiohydrolase gene is obtained froman Aspergillus niger strain. In another preferred aspect, acellobiohydrolase gene is obtained from an Aspergillus oryzae strain. Inanother preferred aspect, a cellobiohydrolase gene is obtained from anEmericella nidulans strain.

Preferred examples of cellobiohydrolase genes that can be used in theinvention are obtained from Acremonium cellulolyticus (U.S. Pat. No.6,127,160), Agaricus bisporus (Chow et al., 1994, Appl. Environ.Microbiol. 60: 2779-2785; Yague et al., 1997, Microbiology (Reading,Engl.) 143: 239-244), Aspergillus aculeatus (Takada et al., 1998, J.Ferment. Bioeng. 85: 1-9), Aspergillus niger (Gielkens et al., 1999,Appl. Environ. Microbiol. 65: 4340-4345), Aspergillus oryzae (Kitamotoet al., 1996, Appl. Microbiol. Biotechnol. 46: 538-544), Athelia rolfsii(EMBL accession number AB103461), Chaetomium thermophilum (EMBLaccession numbers AX657571 and CQ838150), Cullulomonas fimi (Meinke etal., 1994, Mol. Microbiol. 12: 413-422), Emericella nidulans (Lockingtonet al., 2002, Fungal Genet. Biol. 37: 190-196), Fusarium oxysporum(Hagen et al., 1994, Gene 150: 163-167), Geotrichum sp. 128 (EMBLaccession number AB089343), Humicola grisea (de Oliviera and Radford,1990, Nucleic Acids Res. 18: 668; Takashima et al., 1998, J. Biochem.124: 717-725), Humicola nigrescens (EMBL accession number AX657571),Hypocrea koningii (Teeri et al., 1987, Gene 51: 43-52), Myceliopterathermophila (EMBL accession numbers AX657599), Neocallimastixpatriciarum (Denman et al., 1996, Appl. Environ. Microbiol. 62 (6),1889-1896), Phanerochaete chrysosporium (Tempelaars et al., 1994, Appl.Environ. Microbiol. 60: 4387-4393), Thermobifida fusca (Zhang, 1995,Biochemistry 34: 3386-3395), Trichoderma reesei (Terri to al., 1983,Bio/Technology 1: 696-699; Chen et al., 1987, Bio/Technology 5:274-278), and Trichoderma viride (EMBL accession numbers A4368686 andA4368688).

Beta-Glucosidase

Beta-glucosidase, a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21),catalyzes the hydrolysis of terminal non-reducing beta-D-glucoseresidues with the release of beta-D-glucose. For purposes of the presentinvention, beta-glucosidase activity is determined according to thebasic procedure described by Venturi et al., 2002, J. Basic Microbiol.42: 55-66, except different conditions were employed as describedherein. One unit of beta-glucosidase activity is defined as 1.0 μmole ofp-nitrophenol produced per minute at 50° C., pH 5 from 4 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodiumcitrate, 0.01% Tween-20.

Encompassed within the definition of beta-glucosidases are cellobiases.Cellobiases hydrolyze cellobiose to glucose.

In a preferred aspect, a beta-glucosidase gene is obtained from anAspergillus aculeatus strain. In another preferred aspect, abeta-glucosidase gene is obtained from an Aspergillus kawachi strain. Inanother preferred aspect, a beta-glucosidase gene is obtained from aTrichoderma reesei strain.

Preferred examples of beta-glucosidase genes that can be used in theinvention are obtained from Aspergillus aculeatus (Kawaguchi et al.,1996, Gene 173: 287-288), Aspergillus kawachi (Iwashita et al., 1999,Appl. Environ. Microbiol. 65: 5546-5553), Aspergillus oryzae (WO2002/095014), Cellulomonas biazotea (Wong et al., 1998, Gene 207:79-86), Penicillium funiculosum (WO 200478919), Saccharomycopsisfibuligera (Machida et al., 1988, Appl. Environ. Microbiol. 54:3147-3155), Schizosaccharomyces pombe (Wood et al., 2002, Nature 415:871-880), and Trichoderma reesei (Barnett et al., 1991, Bio/Technology9: 562-567).

Glucohydrolases

Glucohydrolase, an exo-1,4-beta-D-glucan glucohydrolase (E.C. 3.2.1.74),catalyzes the hydrolysis of 1,4-linkages (O-glycosyl bonds) in1,4-beta-D-glucans so as to remove successive glucose units. Forpurposes of the present invention, exoglucanase activity is determinedaccording to the procedure described by Himmel et al., 1986, J. Biol.Chem. 261: 12948-12955.

In a preferred aspect, a glucohydrolase gene is obtained from aTrichoderma reesei strain. In another preferred aspect, a glucohydrolasegene is obtained from a Humicola insolens strain. In another preferredaspect, a glucohydrolase gene is obtained from an Aspergillus nigerstrain. In another preferred aspect, a cellobiohydrolase gene isobtained from a Chaetomium thermophilum strain. In another preferredaspect, a glucohydrolase gene is obtained from a Thermoascus aurantiacusstrain. In another preferred aspect, a glucohydrolase gene is obtainedfrom a Thielavia terrestris strain.

Hemicellulases

Enzymatic hydrolysis of hemicellulose can be performed by a wide varietyof fungi and bacteria (Saha, 2003, J. Ind. Microbiol. Biotechnol. 30:279-291). Similar to cellulose degradation, hemicellulose hydrolysisrequires coordinated action of several enzymes. Hemicellulases can beplaced into three general categories: the endo-acting enzymes thatattack internal bonds within the polysaccharide chain, the exo-actingenzymes that act processively from either the reducing or nonreducingend of polysaccharide chain, and the accessory enzymes, acetylesterasesand esterases that hydrolyze lignin glycoside bonds, such as coumaricacid esterase and ferulic acid esterase (Wong, K. K. Y., Tan, L. U. L.,and Saddler, J. N., 1988, Multiplicity of β-1,4-xylanase inmicroorganisms: Functions and applications, Microbiol. Rev., 52:305-317; Tenkanen, M., and Poutanen, K., 1992, Significance of esterasesin the degradation of xylans, in Xylans and Xylanases, Visser, J.,Beldman, G., Kuster-van Someren, M. A., and Voragen, A. G. J., eds.,Elsevier, New York, N.Y., 203-212; Coughlan, M. P., and Hazlewood, G.P., 1993, Hemicellulose and hemicellulases, Portland, London, UK;Brigham, J. S., Adney, W. S., and Himmel, M. E., 1996, Hemicellulases:Diversity and applications, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,119-141).

Examples of endo-acting hemicellulases and accessory enzymes includeendoarabinanase, endoarabinogalactanase, endoglucanase, endomannanase,endoxylanase, and feraxan endoxylanase. Examples of exo-actinghemicellulases and accessory enzymes include α-L-arabinosidase,β-L-arabinosidase, α-1,2-L-fucosidase, α-D-galactosidase,β-D-galactosidase, β-D-glucosidase, β-D-glucuronidase, β-D-mannosidase,β-D-xylosidase, exo-glucosidase, exo-cellobiohydrolase,exo-mannobiohydrolase, exo-mannanase, exo-xylanase, xylanα-glucuronidase, and coniferin β-glucosidase. Examples of esterasesinclude acetyl esterases (acetylgalactan esterase, acetylmannanesterase, and acetylxylan esterase), and aryl esterases (coumaric acidesterase and ferulic acid esterase).

Hemicellulases include xylanases, arabinofuranosidases, acetyl xylanesterases, glucuronidases, endo-galactanases, mannanases, endo- orexo-arabinases, exo-galactanases, and mixtures thereof. Preferably, thehemicellulase is an exo-acting hemicellulase, and more preferably, anexo-acting hemicellulase which has the ability to hydrolyzehemicellulose preferably in the pH range of about 2 to about 7.

A hemicellulase, such as a xylanase, arabinofuranosidase, acetyl xylanesterase, glucuronidase, endo-galactanase, mannanase, endo- orexo-arabinase, or exo-galactanase, or genes thereof, may be obtainedfrom any suitable source, including fungal and bacterial organisms, suchas Aspergillus, Disporotrichum, Penicillium, Neurospora, Fusarium,Trichoderma, Humicola, Thermomyces, and Bacillus.

Preferred examples of hemicellulase genes that can be used in theinvention are obtained from Acidobacterium capsulatum (Inagaki et al.,1998, Biosci. Biotechnol. Biochem. 62: 1061-1067), Agaricus bisporus (DeGroot et al., 1998, J. Mol. Biol. 277: 273-284), Aspergillus aculeatus(U.S. Pat. No. 6,197,564; U.S. Pat. No. 5,693,518), Aspergillus kawachii(Ito et al., 1992, Biosci. Biotechnol. Biochem. 56: 906-912),Aspergillus niger (EMBL accession number AF108944), Magnaporthe grisea(Wu et al., 1995, Mol. Plant. Microbe Interact. 8: 506-514), Penicilliumchrysogenum (Haas et al., 1993, Gene 126: 237-242), Talaromycesemersonii (WO 02/24926), and Trichoderma reesei (EMBL accession numbersX69573, X69574, and AY281369).

Lignin-Degrading Enzymes

Lignin is an aromatic polymer occurring in the woody tissue of higherplants. Due to its hydrophobicity and complex random structure lackingregular hydrolyzable bonds, lignin is poorly degraded by most organisms.The best degraders of lignin are white rot fungi that produceextracellular peroxidases and laccases, which are involved in theinitial attack of lignin.

Lignin-degrading enzymes include, but are not limited to, ligninperoxidases, manganese-dependent peroxidases, hybrid peroxidases, withcombined properties of lignin peroxidases and manganese-dependentperoxidases, and laccases (Vicuna, 2000, supra; Broda et al., 1996,supra). Hydrogen peroxide, required as a co-substrate by theperoxidases, can be generated by glucose oxidase, aryl alcohol oxidase,and/or lignin peroxidase-activated glyoxal oxidase.

Manganese-dependent peroxidase is a frequently encountered peroxidaseproduced by white rot fungi. The peroxidase has a catalytic cycleinvolving a 2-electron oxidation of the heme by hydrogen peroxide andsubsequent oxidation of compound I via compound II in two 1-electronsteps to the native enzyme. The best reducing substrate for compounds Iand II is Mn(II), a metal naturally present in wood. The Mn(III) formedoxidizes other substrates.

Organic acids such as oxalate, glyoxylate, and lactate are known to havean important role in the mechanism of manganese-dependent peroxidase andlignin degradation. Mn(III) is stripped from the enzyme by organicacids, and the produced Mn(III)-organic acid complex acts as adiffusible mediator in the oxidation of lignin by manganese-dependentperoxidase. Mn(III) can also oxidize organic acids, yielding radicals.The organic acids may also be supplied from the degradation of ligninand by microorganisms.

Lignin-degrading enzymes and genes thereof may be obtained from aBjerkandera adusta, Ceriporiopsis subvermispora (see WO 02/079400),Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicolalanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa,Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata,Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametesversicolor, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride strain.

Preferred examples of genes encoding lignin-degrading enzymes that canbe used in the invention are obtained from Bjerkandera adusta (WO2001/098469), Ceriporiopsis subvermispora (Conesa et al., 2002, Journalof Biotechnology 93: 143-158), Cantharellus cibariusi (Ng et al., 2004,Biochemical and Biophysical Research Communications 313: 37-41),Coprinus cinereus (WO 97/008325; Conesa et al., 2002, supra), Lentinulaedodes (Nagai et al., 2002, Applied Microbiology and Biotechnology 60:327-335, 2002), Melanocarpus albomyces (Kiiskinen et al., 2004, FEBSLetters 576: 251-255, 2004), Myceliophthora thermophila (WO 95/006815),Phanerochaete chrysosporium (Conesa et al., 2002, supra; Martinez, 2002,Enzyme and Microbial Technology 30: 425-444, 2002), Phlebia radiata(Conesa et al., 2002, supra), Pleurotus eryngii (Conesa et al., 2002,supra), Polyporus pinsitus (WO 96/000290), Rigidoporus lignosus(Garavaglia et al., 2004, Journal of Molecular Biology 342: 1519-1531),Rhizoctonia solani (WO 96/007988), Scytalidium thermophilum (WO95/033837), Tricholoma giganteum (Wang et al., 2004, Biochemical andBiophysical Research Communications 315: 450-454), and Trametesversicolor (Conesa et al., 2002, supra).

Esterases

Esterase, a carboxylic ester hydrolase (EC 3.1.1), catalyzes thehydrolysis of ester bonds. Esterases useful in the degradation orconversion of plant cell wall polysaccharides include acetyl esterasessuch as acetylgalactan esterase, acetylmannan esterase, and acetylxylanesterase, and esterases that hydrolyze lignin glycoside bonds, such ascoumaric acid esterase and ferulic acid esterase.

Non-limiting examples of esterases include arylesterase, triacylglycerollipase, acetylesterase, acetylcholinesterase, cholinesterase,tropinesterase. pectinesterase, sterol esterase, chlorophyllase,L-arabinonolactonase, gluconolactonase, uronolactonase, tannase,retinyl-palmitate esterase, hydroxybutyrate-dimer hydrolase,acylglycerol lipase, 3-oxoadipate enol-lactonase, 1,4-lactonase,galactolipase, 4-pyridoxolactonase, acylcarnitine hydrolase,aminoacyl-tRNA hydrolase, D-arabinonolactonase,6-phosphogluconolactonase, phospholipase A1, 6-acetylglucosedeacetylase, lipoprotein lipase, dihydrocoumarin lipase,limonin-D-ring-lactonase, steroid-lactonase, triacetate-lactonase,actinomycin lactonase, orsellinate-depside hydrolase, cephalosporin-Cdeacetylase, chlorogenate hydrolase, alpha-amino-acid esterase,4-methyloxaloacetate esterase, carboxymethylenebutenolidase,deoxylimonate A-ring-lactonase, 2-acetyl-1-alkylglycerophosphocholineesterase, fusarinine-C ornithinesterase, sinapine esterase, wax-esterhydrolase, phorbol-diester hydrolase, phosphatidylinositol deacylase,sialate O-acetylesterase, acetoxybutynylbithiophene deacetylase,acetylsalicylate deacetylase, methylumbelliferyl-acetate deacetylase,2-pyrone-4,6-dicarboxylate lactonase, N-acetylgalactosaminoglycandeacetylase, juvenile-hormone esterase, bis(2-ethylhexyl)phthalateesterase, protein-glutamate methylesterase, 11-cis-retinyl-palmitatehydrolase, all-trans-retinyl-palmitate hydrolase,L-rhamnono-1,4-lactonase, 5-(3,4-diacetoxybut-1-ynyl)-2,2′-bithiophenedeacetylase, fatty-acyl-ethyl-ester synthase, xylono-1,4-lactonase,N-acetylglucosaminylphosphatidylinositol deacetylase, cetraxatebenzylesterase, acetylalkylglycerol acetyl hydrolase, and acetylxylanesterase.

Preferred esterases for use in the present invention are lipolyticenzymes, such as, lipases (EC 3.1.1.3, EC 3.1.1.23 and/or EC 3.1.1.26)and phospholipases (EC 3.1.1.4 and/or EC 3.1.1.32, includinglysophospholipases classified by EC 3.1.1.5). Other preferred esterasesare cutinases (EC 3.1.1.74). Further preferred esterases are acetylxylanesterase and pectin methylesterase.

The esterase may be added in an amount effective to obtain the desiredbenefit to improve the performance of the spent whole broth or afermenting microorganism, e.g., to change the lipidcomposition/concentration inside and/or outside of the fermentingmicroorganism or in the cell membrane of the fermenting microorganism,to result in an improvement in the movement of solutes into and/or outof the fermenting microorganisms during fermentation and/or to providemore metabolizable energy sources (such as, e.g., by convertingcomponents, such as, oil from the corn substrate, to components usefulthe fermenting microorganism, e.g., unsaturated fatty acids andglycerol), to increase ethanol yield. Examples of effective amounts ofesterase are from 0.01 to 400 LU/g DS (Dry Solids). Preferably, theesterase is used in an amount of 0.1 to 100 LU/g DS, more preferably 0.5to 50 LU/g DS, and even more preferably 1 to 20 LU/g DS. Furtheroptimization of the amount of esterase can hereafter be obtained usingstandard procedures known in the art.

One Lipase Unit (LU) is the amount of enzyme which liberates 1.0 μmol oftitratable fatty acid per minute with tributyrin as substrate and gumarabic as an emulsifier at 30° C., pH 7.0 (phosphate buffer).

In a preferred aspect the esterase is a lipolytic enzyme, morepreferably, a lipase. As used herein, a “lipolytic enzyme” refers tolipases and phospholipases (including lyso-phospholipases). In a morepreferred aspect, the lipolytic enzyme is a lipase. Lipases may beapplied herein for their ability to modify the structure and compositionof triglyceride oils and fats in the fermentation media (includingfermentation yeast), for example, resulting from a corn substrate.Lipases catalyze different types of triglyceride conversions, such ashydrolysis, esterification and transesterification. Suitable lipasesinclude acidic, neutral and basic lipases, as are well-known in the art,although acidic lipases (such as, e.g., the lipase G AMANO 50, availablefrom Amano) appear to be more effective at lower concentrations oflipase as compared to either neutral or basic lipases. Preferred lipasesfor use in the present invention included Candida antarctica lipase andCandida cylindracea lipase. More preferred lipases are purified lipasessuch as Candida antarctica lipase (lipase A), Candida antarctica lipase(lipase B), Candida cylindracea lipase, and Penicillium camembertiilipase.

The lipase may be the lipase disclosed in EP 258,068-A or may be alipase variant such as a variant disclosed in WO 00/60063 or WO00/32758, hereby incorporated by reference.

Lipases are preferably present in amounts from about 1 to 400 LU/g DS,preferably 1 to 10 LU/g DS, and more preferably 1 to 5 LU/g DS.

The lipolytic enzyme is preferably of microbial origin, in particular,of bacterial, fungal or yeast origin. The lipolytic enzyme or genethereof used may be obtained from any source, including, for example, astrain of Absidia, in particular Absidia blakesleena and Absidiacorymbifera, a strain of Achromobacter, in particular Achromobacteriophagus, a strain of Aeromonas, a strain of Alternaria, in particularAlternaria brassiciola, a strain of Aspergillus, in particularAspergillus niger and Aspergillus flavus, a strain of Achromobacter, inparticular Achromobacter iophagus, a strain of Aureobasidium, inparticular Aureobasidium pullulans, a strain of Bacillus, in particularBacillus pumilus, Bacillus strearothermophilus, and Bacillus subtilis, astrain of Beauveria, a strain of Brochothrix, in particular Brochothrixthermosohata, a strain of Candida, in particular Candida cylindracea(Candida rugosa), Candida paralipolytica, and Candida antarctica, astrain of Chromobacter, in particular Chromobacter viscosum, a strain ofCoprinus, in particular Coprinus cinerius, a strain of Fusarium, inparticular Fusarium oxysporum, Fusarium solani, Fusarium solani pisi,Fusarium roseum culmorum, and Fusarium venenatum, a strain of Geotricum,in particular Geotricum penicillatum, a strain of Hansenula, inparticular Hansenula anomala, a strain of Humicola, in particularHumicola brevispora, Humicola brevis var. thermoidea, and Humicolainsolens, a strain of Hyphozyma, a strain of Lactobacillus, inparticular Lactobacillus curvatus, a strain of Metarhizium, a strain ofMucor, a strain of Paecilomyces, a strain of Penicillium, in particularPenicillium cyclopium, Penicillium crustosum and Penicillium expansum, astrain of Pseudomonas in particular Pseudomonas aeruginosa, Pseudomonasalcaligenes, Pseudomonas cepacia (syn. Burkholderia cepacia),Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas maltophilia,Pseudomonas mendocina, Pseudomonas mephitica lipolytica, Pseudomonasalcaligenes, Pseudomonas plantari, Pseudomonas pseudoalcaligenes,Pseudomonas putida, Pseudomonas stutzeri, and Pseudomonaswisconsinensis, a strain of Rhizoctonia, in particular Rhizoctoniasolani, a strain of Rhizomucor, in particular Rhizomucor miehei, astrain of Rhizopus, in particular Rhizopus japonicus, Rhizopusmicrosporus, and Rhizopus nodosus, a strain of Rhodosporidium, inparticular Rhodosporidium toruloides, a strain of Rhodotorula, inparticular Rhodotorula glutinis, a strain of Sporobolomyces, inparticular Sporobolomyces shibatanus, a strain of Thermomyces, inparticular Thermomyces lanuginosus (formerly Humicola lanuginosa), astrain of Thiarosporella, in particular Thiarosporella phaseolina, astrain of Trichoderma, in particular, Trichoderma harzianum andTrichoderma reesei, and/or a strain of Verticillium.

In a preferred aspect, the lipolytic enzyme or gene thereof is obtainedfrom a strain of Aspergillus, Achromobacter, Bacillus, Candida,Chromobacter, Fusarium, Humicola, Hyphozyma, Pseudomonas, Rhizomucor,Rhizopus, or Thermomyces.

Preferred examples of lipase genes that can be used in the invention areobtained from Absidia sp. (WO 97/027276), Candida antarctica (EMBLaccession number Z30645), Candida cylindracea (EMBL accession numbersX64703, X64704, X66006, X66007, and X66008), Fusarium oxysporum (WO98/26057), Penicillium camembertii (Yamaguchi et al., 1991, Gene 103:61-67), and Thermomyces lanuginosus (EMBL accession number AF054513).

In another preferred aspect, at least one esterase is a cutinase.Cutinases are enzymes which are able to degrade cutin. The cutinase orgene thereof may be obtained from any source. In a preferred aspect, thecutinase or gene thereof is obtained from a strain of Aspergillus, inparticular Aspergillus oryzae, a strain of Alternaria, in particularAlternaria brassiciola, a strain of Fusarium, in particular Fusariumsolani, Fusarium solani pisi, Fusarium roseum culmorum, or Fusariumroseum sambucium, a strain of Helminthosporum, in particularHelminthosporum sativum, a strain of Humicola, in particular Humicolainsolens, a strain of Pseudomonas, in particular Pseudomonas mendocinaor Pseudomonas putida, a strain of Rhizoctonia, in particularRhizoctonia solani, a strain of Streptomyces, in particular Streptomycesscabies, or a strain of Ulocladium, in particular Ulocladiumconsortiale.

In a most preferred aspect, the cutinase or gene thereof is obtainedfrom a strain of Humicola insolens, in particular Humicola insolens DSM1800. Humicola insolens cutinase is described in WO 96/13580 which ishereby incorporated by reference. The cutinase gene may encode a variantsuch as one of the variants disclosed in WO 00/34450 and WO 01/92502,hereby incorporated by reference. Preferred cutinase variants includevariants listed in Example 2 of WO 01/92502 which are herebyspecifically incorporated by reference. An effective amount of cutinaseis between 0.01 and 400 LU/g DS, preferably from about 0.1 to 100 LU/gDS, more preferably, 1 to 50 LU/g DS.

Preferred examples of cutinase genes that can be used in the inventionare obtained from Fusarium solani (WO 90/09446; U.S. Pat. No. 5,827,719;WO 00/34450; and WO 01/92502) and Humicola insolens (WO 96/13580), andvariants thereof.

In another preferred aspect, at least one esterase is a phospholipase.As used herein, the term “phospholipase” is an enzyme which has activitytowards phospholipids.

Phospholipids, such as lecithin or phosphatidylcholine, consist ofglycerol esterified with two fatty acids in an outer (sn-1) and themiddle (sn-2) positions and esterified with phosphoric acid in the thirdposition. The phosphoric acid, in turn, may be esterified to anamino-alcohol. Several types of phospholipase activity can bedistinguished, including phospholipases A₁ and A₂ which hydrolyze onefatty acyl group (in the sn-1 and sn-2 position, respectively) to formlysophospholipid; and lysophospholipase (or phospholipase B), whichhydrolyze the remaining fatty acyl group in lysophospholipid.Phospholipase C and phospholipase D (phosphodiesterases) release diacylglycerol or phosphatidic acid, respectively.

The term “phospholipase” includes enzymes with phospholipase activity,e.g., phospholipase A (A₁ or A₂), phospholipase B activity,phospholipase C activity, or phospholipase D activity. The phospholipaseactivity may be provided by enzymes having other activities as well,such as, e.g., a lipase with phospholipase activity. In other aspects ofthe invention, phospholipase activity is provided by an enzyme havingessentially only phospholipase activity and wherein the phospholipaseenzyme activity is not a side activity.

The phospholipase or gene thereof may be of any origin, e.g., of animalorigin (e.g., mammalian such as from bovine or porcine pancreas), orsnake venom or bee venom. Alternatively, the phospholipase may be ofmicrobial origin, e.g., from filamentous fungi, yeast, or bacteria, suchas Aspergillus, e.g., Aspergillus fumigatus, Aspergillus awamori,Aspergillus foetidus, Aspergillus japonicus, Aspergillus niger, andAspergillus oryzae, Dictyostelium, e.g., Dictyostelium discoideum;Fusarium, e.g., Fusarium culmorum, Fusarium heterosporum, Fusariumoxysporum, Fusarium solani, and Fusarium venenatum; Mucor, e.g., Mucorjaponicus, Mucor mucedo, and Mucor subtilissimus; Neurospora, e.g.,Neurospora crassa; Rhizomucor, e.g., Rhizomucor pusillus; Rhizopus,e.g., Rhizopus arrhizus, Rhizopus japonicus, and Rhizopus stolonifer,Sclerotinia, e.g., Sclerotinia libertiana; Trichophyton, e.g.,Trichophyton rubrum; Whetzelinia, e.g., Whetzelinia sclerotiorum;Bacillus, e.g., Bacillus megaterium and Bacillus subtilis; Citrobacter,e.g., Citrobacter freundii; Enterobacter, e.g., Enterobacter aerogenesand Enterobacter cloacae; Edwardsiella, Edwardsiella tarda; Erwinia,e.g., Erwinia herbicola; Escherichia, e.g., E. coli; Klebsiella, e.g.,Klebsiella pneumoniae; Proteus, e.g., Proteus vulgaris; Providencia,e.g., Providencia stuartii; Salmonella, e.g., Salmonella typhimurium;Serratia, e.g., Serratia liquefasciens and Serratia marcescens;Shigella, e.g., Shigella flexneri; Streptomyces, e.g., Streptomycesvioleceoruber; and Yersinia, e.g., Yersinia enterocolitica. Preferredcommercial phospholipases include LECITASE™ and LECITASE™ ULTRA(available from Novozymes A/S, Denmark).

An effective amount of phospholipase is between 0.01 and 400 LU/g DS,preferably from about 0.1 to 100 LU/g DS, more preferably, 1 to 50 LU/gDS. Further optimization of the amount of phospholipase can hereafter beobtained using standard procedures known in the art.

Enzyme assays for phospholipases are well known in the art (see, forexample, Kim et al., 1997, Anal. Biochem. 250: 109-116; Wu and Cho,1994, Anal. Biochem. 221: 152-159; Hirashima et al., 1983, Brain andNerve 35: 811-817; and Chen et al., 1997, Infection and Immun. 65:405-411).

Preferred examples of phospholipase genes that can be used in theinvention are obtained from Fusarium venenatum (WO 00/028044),Aspergillus oryzae (WO 01/029222), Fusarium oxysporum (WO 98/26057),Penicillum notatum (Masuda et al., 1991, European Journal ofBiochemistry 202: 783-787), Torulaspora delbrueckii (Watanabe et al.,1994, FEMS Microbiology Letters 124: 29-34), Saccharomyces cerevisiae(Lee at al., 1994, Journal of Biological Chemistry 269: 19725-19730),Aspergillus (JP 10155493), Neurospora crassa (EMBL 042791), andSchizosaccharomyces pombe (EMBL 013857).

Proteases

In another preferred aspect, a protease may be useful in the degradationof plant cell wall polysaccharides into one or more products. Theprotease may be used, for example, to digest protein to produce freeamino nitrogen (FAN), where such free amino acids function as nutrientsfor yeast, thereby enhancing the growth of the yeast and, consequently,the production of ethanol. Proteases may also liberate boundpolysaccharide material.

The propagation of a fermenting microorganism with an effective amountof at least one protease may reduce the lag time of the fermentingmicroorganism. The action of the protease in the propagation process isbelieved to directly or indirectly result in the suppression orexpression of genes which are detrimental or beneficial, respectively,to the fermenting microorganism during fermentation, thereby decreasinglag time and resulting in a faster fermentation cycle.

Proteases are well known in the art and refer to enzymes that catalyzethe cleavage of peptide bonds. Suitable proteases include fungal andbacterial proteases. Preferred proteases are acidic proteases, i.e.,proteases characterized by the ability to hydrolyze proteins underacidic conditions below pH 7. Acid fungal proteases or genes thereof canbe obtained from Aspergillus, Mucor, Rhizopus, Candida, Coriolus,Endothia, Enthomophtra, Irpex, Penicillium, Sclerotium, and Torulopsis.In a preferred aspect, a protease or gene thereof is obtained from

Preferably, the protease is an aspartic acid protease, as described, forexample, in Handbook of Proteolytic Enzymes, Edited by A. J. Barrett, N.D. Rawlings and J. F. Woessner, Academic Press, San Diego, 1998, Chapter270).

Enzyme assays for acid proteases, e.g., aspartic acid proteases, arewell known in the art (see, for example, Litvinov et al., 1998, Bioorg.Khim. 24: 175-178).

Preferred examples of acid protease genes that can be used in theinvention are obtained from Aspergillus awamori (Berka et al., 1990,Gene 86: 153-162), Aspergillus niger (Koaze et al., 1964, Agr. Biol.Chem. Japan 28: 216), Aspergillus saitoi (Yoshida, 1954, J. Agr. Chem.Soc. Japan 28: 66), Aspergillus awamori (Hayashida et al., 1977, Agric.Biol. Chem. 42: 927-933), Aspergillus aculeatus (WO 95/02044), andAspergillus oryzae (Berka et al., 1993, Gene 125: 195-198).

Peroxidases

A peroxidase may be any peroxidase (e.g., EC 1.11.1.7), or any fragmentobtained therefrom, exhibiting peroxidase activity.

The peroxidase or gene thereof can be obtained from plants (e.g.,horseradish or soybean peroxidase) or microorganisms (e.g., fungi orbacteria).

Some preferred fungi include strains belonging to the subdivisionDeuteromycotina, class Hyphomycetes, e.g., Fusarium, Humicola,Tricoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces,Ulocladium, Embeffisia, Cladosporium or Dreschlera, in particularFusarium oxysporum (DSM 2672), Humicola insolens, Trichoderma resii,Myrothecium verrucaria ORD 6113), Verticillum alboatrum, Verticillumdahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago,Ulocladium chartarum, Embellisia affi, and Dreschlera halodes.

Other preferred fungi include strains belonging to the subdivisionBasidiomycotina, class Basidiomycetes, e.g., Coprinus, Phanerochaete,Coriolus or Trametes, in particular Coprinus cinereus f. microsporus(IFO 8371), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g.NA-12), or Trametes (previously called Polyporus), e.g., T. versicolor(e.g. PR428-A).

Further preferred fungi include strains belonging to the subdivisionZygomycotina, class Mycoraceae, e.g., Rhizopus or Mucor, in particularMucor hiemalis.

Some preferred bacteria include strains of the order Actinomycetales,e.g. Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus(I FO 12382), and Streptoverticillum verticillium ssp. verticillium.

Other preferred bacteria include Rhodobacter sphaeroides, Rhodomonaspalustri, Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958),Pseudomonas fluorescens (NRRL B-11), and Bacillus strains, e.g.,Bacillus pumilus (ATCC 12905) and Bacillus stearothermophilus.

Further preferred bacteria include strains belonging to Myxococcus,e.g., M. virescens.

In a preferred aspect, a gene encoding a peroxidase is obtained from aCoprinus sp., in particular, Coprinus macrorhizus or Coprinus cinereusaccording to WO 92/16634.

In the present invention, genes encoding a peroxidase includeperoxidases and peroxidase active fragments obtained from cytochromes,haemoglobin, or peroxidase enzymes.

One peroxidase unit (PDXU) is the amount of enzyme which under thefollowing conditions catalyzes the conversion of 1 μmole hydrogenperoxide per minute: 0.1 M phosphate buffer pH 7.0, 0.88 mM hydrogenperoxide, and 1.67 mM 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate)(ABTS) at 30° C. The reaction is followed for 60 seconds (15 secondsafter mixing) by the change in absorbance at 418 nm, which should be inthe range 0.15 to 0.30. For calculation of activity is used anabsorption coefficient of oxidized ABTS of 36 mM⁻¹ cm⁻¹ and astoichiometry of one μmole H₂O₂ converted per two pmole ABTS oxidized.

Preferred examples of peroxidase genes that can be used in the inventionare obtained from Bjerkandera adusta (WO 2001/098469), Ceriporiopsissubvermispora (Conesa et al., 2002, Journal of Biotechnology 93:143-158), Coprinus cinereus (Conesa et al., 2002, supra), Phanerochaetechrysosporium (Conesa et al., 2002, supra), Phlebia radiata (Conesa etal., 2002, supra), Pleurotus eryngii (Conesa et al., 2002, supra), andTrametes versicolor (Conesa et al., 2002, supra).

Laccases

In the present invention, the laccase may be any laccase orlaccase-related enzyme including any laccase (EC 1.10.3.2), any catecholoxidase (EC 1.10.3.1), any bilirubin oxidase (EC 1.3.3.5), or anymonophenol monooxygenase (EC 1.14.18.1).

The above-mentioned enzymes or genes thereof may be obtained from amicroorganism, i.e., bacteria or fungi (including filamentous fungi andyeasts), or they may be obtained from plants.

Suitable fungal sources include Aspergillus, Neurospora, e.g.,Neurospora crassa, Podospora, Botrytis, Collybia, Fomes, Lentinus,Pleurotus, Trametes, e.g., Trametes villosa and Trametes versicolor,Rhizoctonia, e.g., Rhizoctonia solani, Coprinus, e.g., Coprinuscinereus, Coprinus comatus, Coprinus friesii, and Coprinus plicatilis,Psathyrella, e.g., Psathyrella condelleana, Panaeolus, e.g., Panaeoluspapilionaceus, Myceliophthora, e.g., Myceliophthora thermophila,Scytalidium, e.g., Scytalidium thermophilum, Polyporus, e.g., Polyporuspinsitus, Pycnoporus, e.g., Pycnoporus cinnabarinus, Phlebia, e.g.,Phlebia radita (WO 92/01046), or Coriolus, e.g., Coriolus hirsutus (JP2-238885). Suitable bacteria sources are Bacillus.

A laccase or gene thereof is preferably obtained from Coprinus,Myceliophthora, Polyporus, Pycnoporus, Scytalidium or Rhizoctonia; inparticular Coprinus cinereus, Myceliophthora thermophila, Polyporuspinsitus, Pycnoporus cinnabarinus, Scytalidium thermophilum, orRhizoctonia solani.

Laccase activity (LACU) is determined from the oxidation ofsyringaldazine under aerobic conditions. The violet colour produced isphotometered at 530 nm. The analytical conditions are 19 mMsyringaldazine, 23 mM acetate buffer, pH 5.5, 30° C., 1 minute reactiontime. One laccase unit (LACU) is the amount of enzyme that catalyses theconversion of 1.0 μmole syringaldazine per minute at these conditions.

Laccase activity (LAMU) is determined from the oxidation ofsyringaldazine under aerobic conditions. The violet colour produced isphotometered at 530 nm. The analytical conditions are 19 mMsyringaldazine, 23 mM Tris/maleate pH 7.5, 30° C., 1 minute reactiontime. One laccase unit (LAMU) is the amount of enzyme that catalyses theconversion of 1.0 μmole syringaldazine per minute at these conditions.

Preferred examples of laccase genes that can be used in the inventionare obtained from Cantharellus cibariusi (Ng et al., 2004, Biochemicaland Biophysical Research Communications 313: 37-41), Coprinus cinereus(WO 97/008325), Lentinula edodes (Nagai et al., 2002, AppliedMicrobiology and Biotechnology 60: 327-335, 2002), Melanocarpusalbomyces (Kiiskinen et al., 2004, FEBS Letters 576: 251-255, 2004),Myceliophthora thermophila (WO 95/006815), Polyporus pinsitus (WO96/000290), Rigidoporus lignosus (Garavaglia et al., 2004, Journal ofMolecular Biology 342: 1519-1531), Rhizoctonia solani (WO 96/007988),Scytalidium thermophilum (WO 95/033837), and Tricholoma giganteum (Wanget al., 2004, Biochemical and Biophysical Research Communications 315:450-454).

Nucleic Acid Constructs

An isolated gene encoding a plant cell wall polysaccharide degrading orconverting enzyme, e.g., a cellulose-degrading enzyme, hemicellulase,esterase, laccase, ligninase, protease, or peroxidase may be manipulatedin a variety of ways to provide for expression of the enzyme.Manipulation of the gene prior to its insertion into a vector may bedesirable or necessary depending on the expression vector. Thetechniques for modifying nucleotide sequences utilizing recombinant DNAmethods are well known in the art.

The term “nucleic acid construct” as used herein refers to a nucleicacid molecule, either single- or double-stranded, which is isolated froma naturally occurring gene or which has been modified to containsegments of nucleic acids in a manner that would not otherwise exist innature. The term nucleic acid construct is synonymous with the term“expression cassette” when the nucleic acid construct contains thecontrol sequences required for expression of a coding sequence of thepresent invention.

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression of apolypeptide having an enzyme activity of interest. Each control sequencemay be native or foreign to the nucleotide sequence encoding thepolypeptide. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleotide sequence encoding a polypeptide.

The term “operably linked” as used herein refers to a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of the DNA sequence such that the controlsequence directs the expression of a polypeptide.

When used herein the term “coding sequence” is intended to cover anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG. The codingsequence typically include DNA, cDNA, and recombinant nucleotidesequences.

The term “expression” includes any step involved in the production ofthe polypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host for expression of thegene. The promoter sequence contains transcriptional control sequenceswhich mediate the expression of the polypeptide. The promoter may be anynucleotide sequence which shows transcriptional activity in the host ofchoice including mutant, truncated, and hybrid promoters, and may beobtained from genes encoding extracellular or intracellular polypeptideseither homologous or heterologous to the host.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (gIaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionine (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast hosts aredescribed by Romanos et al., 1992, Yeast 8: 423-488.

In the case of the degradation or conversion of plant cell wallpolysaccharides, the choice of the promoter necessarily requires that itbe induced by growth of the host on the polysaccharide biomass.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host to terminate transcription.The terminator sequence is operably linked to the 3′ terminus of thegene encoding an enzyme. Any terminator which is functional in the hostof choice may be used in the present invention.

Preferred terminators for filamentous fungal hosts are obtained from thegenes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Trichodermareesei CBHI, Aspergillus niger alpha-glucosidase, and Fusarium oxysporumtrypsin-like protease.

Preferred terminators for yeast hosts are obtained from the genes forSaccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C(CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphatedehydrogenase. Other useful terminators for yeast hosts are described byRomanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host. The leader sequence is operably linked to the 5′ terminus of agene. Any leader sequence that is functional in the host of choice maybe used in the present invention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of a gene and which, whentranscribed, is recognized by the host as a signal to add polyadenosineresidues to transcribed mRNA. Any polyadenylation sequence which isfunctional in the host of choice may be used in the present invention.

Preferred polyadenylation sequences for filamentous fungal hosts areobtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillusniger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusariumoxysporum trypsin-like protease, and Aspergillus nigeralpha-glucosidase.

Useful polyadenylation sequences for yeast hosts are described by Guoand Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of anenzyme and directs the encoded enzyme into the cell's secretory pathway.The 5′ end of the coding sequence of the gene may inherently contain asignal peptide coding region naturally linked in translation readingframe with the segment of the coding region which encodes the secretedpolypeptide. Alternatively, the 5′ end of the coding sequence maycontain a signal peptide coding region which is foreign to the codingsequence. The foreign signal peptide coding region may be required wherethe coding sequence does not naturally contain a signal peptide codingregion. Alternatively, the foreign signal peptide coding region maysimply replace the natural signal peptide coding region in order toenhance secretion of the enzyme. However, any signal peptide codingregion which directs the expressed polypeptide into the secretorypathway of a host cell of choice, i.e., secreted into a culture medium,may be used in the present invention.

Effective signal peptide coding regions for filamentous fungal hosts arethe signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, Humicola lanuginosa lipase, Trichodermareesei CBHI, Trichoderma reesei CBHII, Trichoderma reesei EGI, andTrichoderma reesei CBHII.

Useful signal peptides for yeast hosts are obtained from the genes forSaccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of anenzyme. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active enzyme bycatalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from genesfor Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei asparticproteinase, and Myceliophthora thermophila laccase (WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of an enzyme, the propeptide region is positioned next tothe amino terminus of the enzyme and the signal peptide region ispositioned next to the amino terminus of the propeptide region.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of an enzyme relative to the growth of thehost. Examples of regulatory systems are those which cause theexpression of a gene to be turned on or off in response to a chemical orphysical stimulus, including the presence of a regulatory compound. Inyeast, the ADH2 system or GAL1 system may be used. In filamentous fungi,the TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter may be used asregulatory sequences. Other examples of regulatory sequences are thosewhich allow for gene amplification. In eukaryotic systems, these includethe dihydrofolate reductase gene which is amplified in the presence ofmethotrexate, and the metallothionein genes which are amplified withheavy metals. In these cases, the gene would be operably linked with theregulatory sequence.

Expression Vectors

The various nucleic acids and control sequences described above may bejoined together to produce a recombinant expression vector which mayinclude one or more convenient restriction sites to allow for insertionor substitution of a gene at such sites. Alternatively, a gene may beexpressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The term “expression vector” encompasses a DNA molecule, linear orcircular, that comprises a segment encoding an enzyme, and which isoperably linked to additional segments that provide for itstranscription.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of a gene of interest. The choice ofthe vector will typically depend on the compatibility of the vector withthe host into which the vector is to be introduced. The vectors may belinear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids which togethercontain the total DNA to be introduced into the genome of the host, or atransposon may be used.

The vectors preferably contain one or more selectable markers whichpermit easy selection of transformed hosts. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like.

Suitable markers for yeast hosts are ADE2, HIS3, LEU2, LYS2, MET3, TRP1,and URA3. Selectable markers for use in a filamentous fungal hostinclude, but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in Aspergillus are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus. Preferred for use in Trichoderma are bar andamdS.

The vectors preferably contain an element(s) that permits integration ofthe vector into the host's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host genome, the vector may rely on the gene'ssequence or any other element of the vector for integration of thevector into the genome by homologous or nonhomologous recombination.Alternatively, the vector may contain additional nucleotide sequencesfor directing integration by homologous recombination into the genome ofthe host. The additional nucleotide sequences enable the vector to beintegrated into the host genome at a precise location(s) in thechromosome(s). To increase the likelihood of integration at a preciselocation, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which are highly homologous with the corresponding targetsequence to enhance the probability of homologous recombination. Theintegrational elements may be any sequence that is homologous with thetarget sequence in the genome of the host. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the host inquestion. The origin of replication may be any plasmid replicatormediating autonomous replication which functions in a cell. The term“origin of replication” or “plasmid replicator” is defined herein as asequence that enables a plasmid or vector to replicate in vivo. Examplesof origins of replication for use in a yeast host are the 2 micronorigin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, andthe combination of ARS4 and CEN6. Examples of origins of replicationuseful in a filamentous fungal cell are AMA1 and ANSI (Gems et al.,1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Research 15:9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction ofplasmids or vectors comprising the gene can be accomplished according tothe methods disclosed in WO 00/24883.

More than one copy of a gene may be inserted into the host to increaseproduction of the gene product. An increase in the copy number of thegene can be obtained by integrating at least one additional copy of thegene into the host genome or by including an amplifiable selectablemarker gene with the nucleotide sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the gene, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Preparation of Spent Whole Fermentation Broth

In the methods of the present invention, the preparation of a spentwhole fermentation broth of a recombinant microorganism can be achievedusing any cultivation method known in the art resulting in theexpression of a plant cell wall polysaccharide degrading or convertingenzyme. Fermentation may, therefore, be understood as comprising shakeflask cultivation, small- or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermenters performed in a suitable medium andunder conditions allowing the cellulase to be expressed or isolated. Theterm “spent whole fermentation broth” is defined herein asunfractionated contents of fermentation material that includes culturemedium, extracellular proteins (e.g., enzymes), and cellular biomass. Itis understood that the term “spent whole fermentation broth” alsoencompasses cellular biomass that has been lysed or permeabilized usingmethods well known in the art.

Generally, the recombinant microorganism is cultivated in a nutrientmedium suitable for production of enzymes having plant cell walldegrading or converting activity. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand cellulase production are known in the art (see, e.g., Bailey, J. E.,and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, NY, 1986).

The enzymes may be detected using methods known in the art that arespecific for the polypeptides, for example, as described supra.

In the methods of the present invention, the spent whole fermentationbroth is preferably used “as is” without any processing or minimaltreatment such as refrigeration to preserve activity, heat treatment toprevent or decrease organism viability, or addition of chemical agentsthat prevent or decrease organism viability.

The cellulose-degrading activity of the spent whole fermentation brothmay be determined using carboxymethyl cellulose (CMC) as a substrate.Hydrolysis of carboxymethyl cellulose (CMC) decreases the viscosity ofthe assay mixture, which may be determined by a vibration viscosimeter(e.g., MIVI 3000 from Sofraser, France). Determination ofcellulose-degrading activity, measured in terms of Cellulase ViscosityUnit (CEVU), quantifies the amount of catalytic activity present in thespent whole fermentation broth by measuring the ability of the sample toreduce the viscosity of a solution of carboxymethyl cellulose (CMC). Theassay is carried out at 40° C.; pH 9.0; 0.1M phosphate buffer; time 30minutes; CMC substrate (33.3 g/L carboxymethyl cellulose Hercules 7LFD); enzyme concentration approx. 3.3-4.2 CEVU/ml. The CEVU activity iscalculated relative to a declared enzyme standard, such as Celluzyme™Standard 17-1194 (obtained from Novozymes A/S, Bagsværd, Denmark).

Other enzyme activities can be measured as described herein.

Supplements

In the methods of the present invention, the spent whole fermentationbroth may be supplemented with one or more enzyme activities notexpressed by the recombinant microorganism to improve the degradation orconversion of plant cell wall polysaccharides.

Preferred additional enzymes include, but are not limited to,endoglucanase (cellulase), cellobiohydrolase, beta-glucosidase,endo-beta-1,3(4)-glucanase, glucohydrolase, xyloglucanase, xylanase,xylosidase, alpha-arabinofuranosidase, alpha-glucuronidase, acetyl xylanesterase, mannanase, mannosidase, alpha-galactosidase, mannan acetylesterase, galactanase, arabinanase, pectate lyase, pectin lyase, pectatelyase, polygalacturonase, pectin acetyl esterase, pectin methylesterase, alpha-arabinofuranosidase, beta-galactosidase, galactanase,arabinanase, alpha-arabinofuranosidase, rhamnogalacturonase,rhamnogalacturonan lyase, rhamnogalacturonan acetyl esterase,xylogalacturonosidase, xylogalacturonase, rhamnogalacturonan lyase,lignin peroxidases, manganese-dependent peroxidases, hybrid peroxidases,with combined properties of lignin peroxidases and manganese-dependentperoxidases, and laccases.

The enzymes may be obtained from a suitable microbial or plant source orby recombinant means as described herein or may be obtained fromcommercial sources.

The additional enzyme(s) added as a supplement to the spent whole brothmay be used “as is” or may be purified. The term “as is” as used hereinrefers to an enzyme preparation produced by fermentation that undergoesno or minimal recovery and/or purification. The term “purified” as usedherein covers enzymes free from other components from the organism fromwhich it is obtained. The term “purified” also covers enzymes free fromcomponents from the native organism from which it is obtained. Theenzymes may be purified, with only minor amounts of other proteins beingpresent. The term “purified” as used herein also refers to removal ofother components, particularly other proteins and most particularlyother enzymes present in the cell of origin of the enzyme. The enzymemay be “substantially pure,” that is, free from other components fromthe organism in which it is produced, that is, for example, a hostorganism for enzymes produced by recombinant means. In preferred aspect,the enzymes are at least 20% pure, preferably at least 40% pure, morepreferably at least 60% pure, more preferably at least 80% pure, evenmore preferably at least 90% pure, most preferably at least 95% pure,and even most preferably at least 99% pure, as determined by SDS-PAGE.

Where the enzyme(s) is obtained from a suitable microbial or plantsource or by recombinant means, the enzyme may be recovered usingrecovery methods well known in the art. For example, the enzyme may berecovered from a nutrient medium by conventional procedures including,but not limited to, centrifugation, filtration, extraction,spray-drying, evaporation, or precipitation.

The enzyme(s) may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989).

The enzymes may also be obtained from commercial sources.

Examples of cellulases suitable for use in the present inventioninclude, for example, CELLUCLAST™ (available from Novozymes A/S),NOVOZYM™ 188 (available from Novozymes A/S). Other commerciallyavailable preparations comprising cellulase which may be used includeCELLUZYME™, CEREFLO™ and ULTRAFLO™ (Novozymes A/S), LAMINEX™ andSPEZYME™ CP (Genencor Int.) and ROHAMENT™ 7069 W (Röhm GmbH). Thecellulase enzymes are added in amounts effective from about 0.001 to5.0% wt. of solids, more preferably from about 0.025% to 4.0% wt. ofsolids, and most preferably from about 0.005% to 2.0% wt. of solids.

Preferred commercially available preparations comprising xylanaseinclude SHEARZYME®, BIOFEED WHEAT®, BIO-FEED Plus® L, CELLUCLAST®,ULTRAFLO®, VISCOZYME®, PENTOPAN MONO® BG, PULPZYME® HC (Novozymes A/S);LAMINEX®, SPEZYME® CP (Genencor Int.). The hemicellulase is preferablyadded in an amount effective of from about 0.001 to 5.0% wt. of solids,more preferably from about 0.025 to 4.0% wt. of solids, and mostpreferably from about 0.005 to 2.0% wt. of solids.

A preferred commercially available preparation comprising hemicellulaseincludes VISCOZYME™ (Novozymes A/S). The hemicellulase enzymes are addedin amounts effective from about 0.001 to 5.0% wt. of solids, morepreferably from about 0.025% to 4.0% wt. of solids, and most preferablyfrom about 0.005% to 2.0% wt. of solids.

Preferred commercial lipases include LECITASE™, LIPOLASE™ and LIPEX™(Novozymes A/S, Denmark) and G AMANO™ 50 (Amano). Lipases are preferablyadded or present in amounts from about 1 to 400 LU/g DS, preferably 1 to10 LU/g DS, and more preferably 1 to 5 LU/g DS.

Preferred commercial phospholipases include LECITASE™ and LECITASE™ULTRA (Novozymes A/S, Denmark).

Preferred commercial proteases include ALCALASE™, SAVINASE™, andNEUTRASE™ (Novozymes A/S), GC106 (Genencor Int, Inc.), and NOVOZYM™50006 (Novozymes A/S).

The additional enzyme(s) used in the present invention may be in anyform suitable for use in the processes described herein, such as, e.g.,in the form of a dry powder or granulate, a non-dusting granulate, aliquid, a stabilized liquid, or a protected enzyme. Granulates may beproduced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452,and may optionally be coated by process known in the art. Liquid enzymepreparations may, for instance, be stabilized by adding stabilizers suchas a sugar, a sugar alcohol or another polyol, lactic acid or anotherorganic acid according to established process. Protected enzymes may beprepared according to the process disclosed in EP 238,216.

Processing of Plant Cell Wall Polysaccharides

The methods of the present invention may be used in the production ofmonosaccharides, disaccharides, and polysaccharides as chemical orfermentation feedstocks from biomass for the production of organicproducts, chemicals and fuels, plastics, and other products orintermediates. In particular, the value of processing residues (drieddistillers grain, spent grains from brewing, sugarcane bagasse, etc.)can be increased by partial or complete solubilization of cellulose orhemicellulose. In addition to ethanol, some commodity and specialtychemicals that can be produced from cellulose and hemicellulose includexylose, acetone, acetate, glycine, lysine, organic acids (e.g., lacticacid), 1,3-propanediol, butanediol, glycerol, ethylene glycol, furfural,polyhydroxyalkanoates, cis, cis-muconic acid, and animal feed (Lynd, L.R., Wyman, C. E., and Gerngross, T. U., 1999, Biocommodity engineering,Biotechnol. Prog., 15: 777-793; Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212; and Ryu, D. D. Y., and Mandels, M., 1980, Cellulases:biosynthesis and applications, Enz. Microb. Technol., 2: 91-102).Potential coproduction benefits extend beyond the synthesis of multipleorganic products from fermentable carbohydrate. Lignin-rich residuesremaining after biological processing of a plant cell wallpolysaccharide can be converted to lignin-obtained chemicals, or usedfor power production (Lynd et al., 1999, supra; Philippidis, 1996,supra; Ryu and Mandels, 1980, supra).

Conventional methods used to process the plant cell wall polysaccharidesin accordance with the methods of the present invention are wellunderstood to those skilled in the art. The methods of the presentinvention may be implemented using any conventional biomass processingapparatus configured to operate in accordance with the invention.

Such an apparatus may include, but is not limited to, a batch-stirredreactor, a continuous flow stirred reactor with ultrafiltration, acontinuous plug-flow column reactor (Gusakov, A. V., and Sinitsyn, A.P., 1985, Kinetics of the enzymatic hydrolysis of cellulose: 1. Amathematical model for a batch reactor process, Enz. Microb. Technol.,7: 346-352), an attrition reactor (Ryu, S. K., and Lee, J. M., 1983,Bioconversion of waste cellulose by using an attrition bioreactor,Biotechnol. Bioeng., 25: 53-65), or a reactor with intensive stirringinduced by electromagnetic field (Gusakov, A. V., Sinitsyn, A. P.,Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996, Enhancement ofenzymatic cellulose hydrolysis using a novel type of bioreactor withintensive stirring induced by electromagnetic field, Appl. Biochem.Biotechnol., 56: 141-153).

The conventional methods include, but are not limited to,saccharification, fermentation, separate hydrolysis and fermentation(SHF), simultaneous saccharification and fermentation (SSF),simultaneous saccharification and cofermentation (SSCF), hybridhydrolysis and fermentation (HHF), and direct microbial conversion(DMC).

SHF uses separate process steps to first enzymatically hydrolyzecellulose to glucose and then ferment glucose to ethanol. In SSF, theenzymatic hydrolysis of cellulose and the fermentation of glucose toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF includes the coferementation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog., 15: 817-827).Hybrid hydrolysis and fermentation (HHF) process includes two separatesteps carried out in the same reactor but at different temperatures,high temperature enzymatic saccharification followed by SSF at a lowertemperature that the fermentation strain can tolerate. DMC combines allthree processes (cellulase production, cellulose hydrolysis, andfermentation) in one step (Lynd, L. R., Weimer, P. J., van Zyl, W. H.,and Pretorius, I. S., 2002, Microbial cellulose utilization:Fundamentals and biotechnology, Microbiol. Mol. Biol. Reviews, 66:506-577).

“Fermentation” or “fermentation process” refers to any fermentationprocess or any process comprising a fermentation step. A fermentationprocess includes, without limitation, fermentation processes used toproduce fermentation products including alcohols (e.g., arabinitol,butanol, ethanol, glycerol, methanol, 1,3-propanediol, sorbitol, andxylitol); organic acids (e.g., acetic acid, adipic acid, ascorbic acid,citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid,glucaric acid, gluconic acid, glucuronic acid, glutaric acid,3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonicacid, oxalic acid, propionic acid, succinic acid, and xylonic acid);ketones (e.g., acetone); amino acids (e.g., aspartic acid, glutamicacid, glycine, lysine, serine, and threonine); and/or gases (e.g.,methane, hydrogen (H₂), carbon dioxide (CO₂), and carbon monoxide (CO)).Fermentation processes also include fermentation processes used in theconsumable alcohol industry (e.g., beer and wine), dairy industry (e.g.,fermented dairy products), leather industry, and tobacco industry.

The present invention also relates to methods for producing one or moreorganic substances, comprising: (a) saccharifying plant cell wallpolysaccharides with an effective amount of a spent whole fermentationbroth of a recombinant microorganism, wherein the recombinantmicroorganism expresses one or more heterologous genes encoding enzymeswhich degrade or convert the plant cell wall polysaccharides intosaccharified material; (b) fermenting the saccharified material of step(a) with one or more fermenting microoganisms; and (c) recovering theone or more organic substances from the fermentation.

The organic substance can be any substance derived from thefermentation. In a preferred aspect, the organic substance is analcohol. It will be understood that the term “alcohol” encompasses anorganic substance that contains one or more hydroxyl moieties. In a morepreferred aspect, the alcohol is arabinitol. In another more preferredaspect, the alcohol is butanol. In another more preferred aspect, thealcohol is ethanol. In another more preferred aspect, the alcohol isglycerol. In another more preferred aspect, the alcohol is methanol. Inanother more preferred aspect, the alcohol is 1,3-propanediol. Inanother more preferred aspect, the alcohol is sorbitol. In another morepreferred aspect, the alcohol is xylitol. See, for example, Gong, C. S.,Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production fromrenewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R., 2002,The biotechnological production of sorbitol, Appl. Microbiol.Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes forfermentative production of xylitol—a sugar substitute, ProcessBiochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H.P., 2003, Production of acetone, butanol and ethanol by Clostridiumbeijerinckii BA101 and in situ recovery by gas stripping, World Journalof Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the organic substance is an organic acid.In another more preferred aspect, the organic acid is acetic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the organic substance is a ketone. It willbe understood that the term “ketone” encompasses an organic substancethat contains one or more ketone moieties. In another more preferredaspect, the ketone is acetone. See, for example, Qureshi and Blaschek,2003, supra.

In another preferred aspect, the organic substance is an aldehyde. Inanother more preferred aspect, the aldehyde is a furfural.

In another preferred aspect, the organic substance is an amino acid. Inanother more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is alanine. In anothermore preferred aspect, the amino acid is arginine. In another morepreferred aspect, the amino acid is asparagine. In another morepreferred aspect, the amino acid is glutamine. In another more preferredaspect, the amino acid is glutamic acid. In another more preferredaspect, the amino acid is glycine. In another more preferred aspect, theamino acid is histidine. In another more preferred aspect, the aminoacid is isoleucine. In another more preferred aspect, the amino acid isleucine. In another more preferred aspect, the amino acid is lysine. Inanother more preferred aspect, the amino acid is methionine. In anothermore preferred aspect, the amino acid is phenylalanine. In another morepreferred aspect, the amino acid is proline. In another more preferredaspect, the amino acid is serine. In another more preferred aspect, theamino acid is threonine. In another more preferred aspect, the aminoacid is tryptophan. In another more preferred aspect, the amino acid istyrosine. In another more preferred aspect, the amino acid is valine.See, for example, Richard, A., and Margaritis, A., 2004, Empiricalmodeling of batch fermentation kinetics for poly(glutamic acid)production and other microbial biopolymers, Biotechnology andBioengineering 87 (4): 501-515.

In another preferred aspect, the organic substance is a gas. In anothermore preferred aspect, the gas is methane (CH₄). In another morepreferred aspect, the gas is hydrogen (H₂). In another more preferredaspect, the gas is carbon dioxide (CO₂). In another more preferredaspect, the gas is carbon monoxide (CO). See, for example, Kataoka, N.,A. Miya, and K. Kiriyama, 1997, Studies on hydrogen production bycontinuous culture system of hydrogen-producing anaerobic bacteria,Water Science and Technology 36 (6-7): 41-47; and Gunaseelan V. N. inBiomass and Bioenergy, Vol. 13 (1-2), pp. 83-114, 1997, Anaerobicdigestion of biomass for methane production: A review.

Production of an organic substance from polysaccharides, such ascellulose, typically requires four major steps. These four steps arepretreatment, enzymatic hydrolysis, fermentation, and recovery.Exemplified below is a process for producing ethanol, but it will beunderstood that similar processes can be used to produce other organicsubstances, for example, the substances described above.

Pretreatment.

In the pretreatment or pre-hydrolysis step, the cellulosic material isheated to break down the lignin and carbohydrate structure to make thecellulose fraction accessible to cellulolytic enzymes. The heating isperformed either directly with steam or in slurry where a catalyst mayalso be added to the material to speed up the reactions. Catalystsinclude strong acids, such as sulfuric acid and SO₂, or alkali, such assodium hydroxide. The purpose of the pre-treatment stage is tofacilitate the penetration of the enzymes and microorganisms. Cellulosicbiomass may also be subject to a hydrothermal steam explosionpre-treatment (See U.S. Patent Application No. 20020164730).

Saccharification.

In the enzymatic hydrolysis step, also known as saccharification,enzymes as described herein are added to the pretreated material toconvert the cellulose fraction to glucose and/or other sugars. Thesaccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions. Asaccharification step may last up to 200 hours. Saccharification may becarried out at temperatures from about 30° C. to about 65° C., inparticular around 50° C., and at a pH in the range between about 4 andabout 5, especially around pH 4.5. To produce glucose that can bemetabolized by yeast, the hydrolysis is typically performed in thepresence of a beta-glucosidase.

Fermentation.

In the fermentation step, sugars, released from the plant cell wallpolysaccharides as a result of the pretreatment and enzymatic hydrolysissteps, are fermented to one or more organic substances, e.g., ethanol,by a fermenting organism, such as yeast, or fermenting organisms. Thefermentation can also be carried out simultaneously with the enzymatichydrolysis in the same vessels, again under controlled pH, temperatureand mixing conditions. When saccharification and fermentation areperformed simultaneously in the same vessel, the process is generallytermed simultaneous saccharification and fermentation or SSF.

Any suitable plant cell wall biomass may be used in a fermentationprocess of the present invention. The plant cell wall biomass isgenerally selected based on the desired fermentation product(s) and theprocess employed, as is well known in the art. Examples of substratessuitable for use in the methods of the present invention, includecellulose-containing materials, such as wood or plant residues or lowmolecular sugars DP₁₋₃ obtained from processed plant cell wallpolysaccharides that can be metabolized by the fermenting microorganism,and which may be supplied by direct addition to the fermentation media.

The term “fermentation medium” will be understood to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism suitable for usein a desired fermentation process. Suitable fermenting microorganismsaccording to the invention are able to ferment, i.e., convert, sugars,such as glucose, xylose, arabinose, mannose, galactose, oroligosaccharides, directly or indirectly into the desired fermentationproduct(s). Examples of fermenting microorganisms include fungalorganisms, such as yeast. Preferred yeast include strains ofSaccharomyces spp., and in particular, Saccharomyces cerevisiae.Commercially available yeast include, e.g., Red Star®/Lesaffre EthanolRed (available from Red Star/Lesaffre, USA) FALI (available fromFleischmann's Yeast, a division of Burns Philp Food Inc., USA),SUPERSTART (available from Alltech), GERT STRAND (available from GertStrand AB, Sweden) and FERMIOL (available from DSM Specialties). Othermicroorganisms may also be used depending the fermentation product(s)desired. These other microorganisms include Gram positive bacteria,e.g., Lactobacillus such as Lactobacillus lactis, Propionibacterium suchas Propionibacterium freudenreichii; Clostridium sp. such as Clostridiumbutyricum, Clostridium beijerinckii, Clostridium diolis, Clostridiumacetobutylicum, and Clostridium thermocellum; Gram negative bacteria,e.g., Zymomonas such as Zymomonas mobilis; and filamentous fungi, e.g.,Rhizopus oryzae.

In a preferred aspect, the yeast is a Saccharomyces sp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae. In another morepreferred aspect, the yeast is Saccharomyces distaticus. In another morepreferred aspect, the yeast is Saccharomyces uvarum. In anotherpreferred aspect, the yeast is a Kluyveromyces. In another morepreferred aspect, the yeast is Kluyveromyces marxianus. In another morepreferred aspect, the yeast is Kluyveromyces fragilis. In anotherpreferred aspect, the yeast is a Candida. In another more preferredaspect, the yeast is Candida pseudotropicalis. In another more preferredaspect, the yeast is Candida brassicae. In another preferred aspect, theyeast is a Clavispora. In another more preferred aspect, the yeast isClavispora lusitaniae. In another more preferred aspect, the yeast isClavispora opuntiae. In another preferred aspect, the yeast is aPachysolen. In another more preferred aspect, the yeast is Pachysolentannophilus. In another preferred aspect, the yeast is a Bretannomyces.In another more preferred aspect, the yeast is Bretannomyces clausenii(Philippidis, G. P., 1996, Cellulose bioconversion technology, inHandbook on Bioethanol: Production and Utilization, Wyman, C. E., ed.,Taylor & Francis, Washington, D.C., 179-212).

Bacteria that can efficiently ferment glucose to ethanol include, forexample, Zymomonas mobilis and Clostridium thermocellum (Philippidis,1996, supra).

It is well known in the art that the organisms described above can alsobe used to produce other organic substances, as described herein.

The cloning of heterologous genes into Saccharomyces cerevisiae (Chen,Z., Ho, N. W. Y., 1993, Cloning and improving the expression of Pichiastipitis xylose reductase gene in Saccharomyces cerevisiae, Appl.Biochem. Biotechnol., 39-40: 135-147; Ho, N. W. Y., Chen, Z, Brainard,A. P., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859), or in bacteria such as Escherichia coli (Beall, D. S.,Ohta, K., Ingram, L. O., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303), Klebsiella oxytoca (Ingram, L. O., Gomes, P. F.,Lai, X., Moniruzzaman, M., Wood, B. E., Yomano, L. P., York, S. W.,1998, Metabolic engineering of bacteria for ethanol production,Biotechnol. Bioeng., 58: 204-214), and Zymomonas mobilis (Zhang, M.,Eddy, C., Deanda, K., Finkelstein, M., and Picataggio, S., 1995,Metabolic engineering of a pentose metabolism pathway in ethanologenicZymomonas mobilis, Science, 267: 240-243; Deanda, K., Zhang, M., Eddy,C., and Picataggio, S., 1996, Development of an arabinose-fermentingZymomonas mobilis strain by metabolic pathway engineering, Appl.Environ. Microbiol, 62: 4465-4470) has led to the construction oforganisms capable of converting hexoses and pentoses to ethanol(cofermentation).

Yeast or other microorganisms are typically added to the hydrolysate andthe fermentation is allowed to proceed for 24-96 hours, such as 35-60hours. The temperature is typically between 26-40° C., in particular atabout 32° C., and at pH 3-6, in particular about pH 4-5.

In a preferred aspect, yeast is applied to the hydrolysate and thefermentation proceeds for 24-96 hours, such as typically 35-60 hours. Inanother preferred aspect, the temperature is generally between 26-40°C., in particular about 32° C., and the pH is generally from pH 3 to 6,preferably about pH 4-5. Yeast cells are preferably applied in amountsof 10⁵ to 10¹², preferably from 10⁷ to 10¹⁰, especially 5×10⁷ viableyeast count per ml of fermentation broth. During the ethanol producingphase the yeast cell count should preferably be in the range from 10⁷ to10¹⁰, especially around 2×10⁸. Further guidance in respect of usingyeast for fermentation can be found in, e.g., “The Alcohol Textbook”(Editors K. Jacques, T. P. Lyons and D. R. Kelsall, NottinghamUniversity Press, United Kingdom 1999), which is hereby incorporated byreference.

The most widely used process in the art is the simultaneoussaccharification and fermentation (SSF) process where there is noholding stage for the saccharification, meaning that the fermentatingmicroorganism and enzyme are added together.

For ethanol production, following the fermentation the mash is distilledto extract the ethanol. The ethanol obtained according to the process ofthe invention may be used as, e.g., fuel ethanol; drinking ethanol,i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator may be used in combination with any of theenzymatic processes described herein to further improve the fermentationprocess, and in particular, the performance of the fermentingmicroorganism, such as, rate enhancement and ethanol yield. A“fermentation stimulator” refers to stimulators for growth of thefermenting microorganisms, in particular, yeast. Preferred fermentationstimulators for growth include vitamins and minerals. Examples ofvitamins include multivitamins, biotin, pantothenate, nicotinic acid,meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid,riboflavin, and Vitamins A, B, C, D, and E. See, e.g., Alfenore et al.,Improving ethanol production and viability of Saccharomyces cerevisiaeby a vitamin feeding strategy during fed-batch process,” Springer-Verlag(2002), which is hereby incorporated by reference. Examples of mineralsinclude minerals and mineral salts that can supply nutrients comprisingP, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.

Recovery.

Following the fermentation, the organic substance of interest isrecovered from the mash by any method known in the art. Such methodsinclude, but are not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, distillation, or extraction. For example, in an ethanolfermentation, the alcohol is separated from the fermented plant cellwall polysaccharides and purified by conventional methods ofdistillation. Ethanol with a purity of up to about 96 vol. % ethanol canbe obtained, which can be used as, e.g., fuel ethanol; drinking ethanol,i.e., potable neutral spirits; or industrial ethanol.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

Strains

Trichoderma reesei (synonym Hypocrea jecorina) RutC30 was used as thesource for cellulase. Trichoderma reesei RutC30 is available from theAmerican Type Culture Collection (ATCC 56765). Trichoderma reeseiSMA135-04 is a recombinant derivative of Trichoderma reesei RutC30 thatharbors multiple copies of the Aspergillus oryzae beta-glucosidase geneexpressed under the transcriptional control of the Trichoderma reeseicbh1 gene promoter.

Example 1 Construction of pAlLo01 Expression Vector

Expression vector pAlLo1 was constructed by modifying pBANe6 (U.S. Pat.No. 6,461,837), which comprises a hybrid of the promoters from the genesfor Aspergillus niger neutral alpha-amylase and Aspergillus oryzaetriose phosphate isomerase (NA2-tpi promoter), Aspergillus nigeramyloglucosidase terminator sequence (AMG terminator), and Aspergillusnidulans acetamidase gene (amdS). All mutagenesis steps were verified bysequencing using Big-Dye™ terminator chemistry (Applied Biosystems,Inc., Foster City, Calif.). Modification of pBANe6 was performed byfirst eliminating three Nco I restriction sites at positions 2051, 2722,and 3397 bp from the amdS selection marker by site-directed mutagenesis.All changes were designed to be “silent” leaving the actual proteinsequence of the amdS gene product unchanged. Removal of these threesites was performed simultaneously with a GeneEditor™ in vitroSite-Directed Mutagenesis Kit (Promega, Madison, Wis.) according to themanufacturer's instructions using the following primers (underlinednucleotide represents the changed base):

AMDS3NcoMut (2050): (SEQ ID NO: 1) 5′-GTGCCCCATGATACGCCTCCGG-3′AMDS2NcoMut (2721): (SEQ ID NO: 2) 5′-GAGTCGTATTTCCAAGGCTCCTGACC-3′AMDS1NcoMut (3396): (SEQ ID NO: 3) 5′-GGAGGCCATGAAGTGGACCAACGG-3′

A plasmid comprising all three expected sequence changes was thensubmitted to site-directed mutagenesis, using a QuickChange™Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.), toeliminate the Nco I restriction site at the end of the AMG terminator atposition 1643. The following primers (underlined nucleotide representsthe changed base) were used for mutagenesis:

Upper Primer to mutagenize the AMG terminator sequence: (SEQ ID NO: 4)5′-CACCGTGAAAGCCATGCTCTTTCCTTCGTGTAGAAGACCAGACAG- 3′ Lower Primer tomutagenize the AMG terminator sequence: (SEQ ID NO: 5)5′-CTGGTCTTCTACACGAAGGAAAGAGCATGGCTTTCACGGTGTCTG- 3′

The last step in the modification of pBANe6 was the addition of a newNco I restriction site at the beginning of the polylinker using aQuickChange™ Site-Directed Mutagenesis Kit and the following primers(underlined nucleotides represent the changed bases) to yield pAlLo1(FIG. 6).

Upper Primer to mutagenize the NA2-tpi promoter: (SEQ ID NO: 6)5′-CTATATACACAACTGGATTTACCATGGGCCCGCGGCCGCAGATC-3′ Lower Primer tomutagenize the NA2-tpi promoter: (SEQ ID NO: 7)5′-GATCTGCGGCCGCGGGCCCATGGTAAATCCAGTTGTGTATATAG-3′

Example 2 Construction of pMJ04 Expression Vector

Expression vector pMJ04 was constructed by PCR amplification of theTrichoderma reesei exocellobiohydrolase 1 gene (cbh1) terminator fromTrichoderma reesei RutC30 genomic DNA using primers 993429 (antisense)and 993428 (sense) shown below. The antisense primer was engineered tohave a PacI site at the 5′-end and a SpeI site at the 3′-end of thesense primer.

Primer 993429 (antisense): 5′-AACGTTAATTAAGGAATCGTTTTGTGTTT-3′ (SEQ IDNO: 8) Primer 993428 (sense): 5′-AGTACTAGTAGCTCCGTGGCGAAAGCCTG-3′ (SEQID NO: 9)

Trichoderma reesei RutC30 genomic DNA was isolated using a DNeasy PlantMaxi Kit (QIAGEN Inc., Valencia, Calif.).

The amplification reactions (50 μl) were composed of 1× ThermoPolReaction Buffer (New England BioLabs, Beverly, Mass.), 0.3 mM dNTPs, 100ng of Trichoderma reesei RutC30 genomic DNA, 0.3 μM primer 993429, 0.3μM primer 993428, and 2 units of Vent polymerase (New England BioLabs,Beverly, Mass.). The reactions were incubated in an EppendorfMastercycler 5333 programmed as follows: 30 cycles each for 30 secondsat 94° C., 30 seconds at 55° C., and 30 seconds at 72° C. (15 minutefinal extension).

The reaction products were isolated on a 1.0% agarose gel using 40 mMTris base-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer where a229 bp product band was excised from the gel and purified using a QIAGENQIAquick Gel Extraction Kit according to the manufacturer'sinstructions.

The resulting PCR fragment was digested with Pac I and Spe I and ligatedinto pAlLo1 digested with the same restriction enzymes using a RapidLigation Kit (Roche, Indianapolis, Ind.), to generate pMJ04 (FIG. 2).

Example 3 Construction of pCaHj568 Expression Vector

Expression plasmid pCaHj568 was constructed from pCaHj170 (U.S. Pat. No.5,763,254) and pMT2188. Plasmid pCaHj170 comprises the Humicola insolensendoglucanase V (EGV) coding region. Plasmid pMT2188 was constructed asfollows: The pUC19 origin of replication was PCR amplified from pCaHj483(WO 98/00529) with primers 142779 and 142780 shown below. Primer 142780introduces a Bbu I site in the PCR fragment.

142779: (SEQ ID NO: 10) 5′-TTGAATTGAAAATAGATTGATTTAAAACTTC-3′ 142780:(SEQ ID NO: 11) 5′-TTGCATGCGTAATCATGGTCATAGC-3′

The Expand PCR System (Roche Molecular Biochemicals, Basel, Switserland)was used for the amplification following the manufacturer's instructionsfor this and the subsequent PCR amplifications. PCR products wereseparated on an agarose gel and an 1160 bp fragment was isolated andpurified using a Jetquick Gel Extraction Spin Kit (Genomed, Wielandstr,Germany).

The URA3 gene was amplified from the general Saccharomyces cerevisiaecloning vector pYES2 (Invitrogen, Carlsbad, Calif.) using primers 140288and 142778 below. Primer 140288 introduces an Eco RI site in the PCRfragment.

140288: (SEQ ID NO: 12) 5′-TTGAATTCATGGGTAATAACTGATAT-3′ 142778: (SEQ IDNO: 13) 5′-AAATCAATCTATTTTCAATTCAATTCATCATT-3′PCR products were separated on an agarose gel and an 1126 bp fragmentwas isolated and purified using a Jetquick Gel Extraction Spin Kit.

The two PCR fragments were fused by mixing and amplifed using primers142780 and 140288 shown above by overlap method splicing (Horton et al.,1989, Gene 77: 61-68). PCR products were separated on an agarose gel anda 2263 bp fragment was isolated and purified using a Jetquick GelExtraction Spin Kit.

The resulting fragment was digested with Eco RI and Bbu I and ligated tothe largest fragment of pCaHj483 digested with the same enzymes. Theligation mixture was used to transform pyrF⁻ E. coli strain DB6507 (ATCC35673) made competent by the method of Mandel and Higa, 1970, J. Mol.Biol. 45: 154. Transformants were selected on solid M9 medium (Sambrooket al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, ColdSpring Harbor Laboratory Press) supplemented per liter with 1 g ofcasamino acids, 500 μg of thiamine, and 10 mg of kanamycin. A plasmidfrom one transformant was isolated and designated pCaHj527 (FIG. 3).

The NA2/tpi promoter present on pCaHj527 was subjected to site-directedmutagenesis by a simple PCR approach. Nucleotides 134-144 were convertedfrom GTACTAAAACC to CCGTTAAATTT using mutagenic primer 141223:

Primer 141223: (SEQ ID NO: 14)5′-GGATGCTGTTGACTCCGGAAATTTAACGGTTTGGTCTTGCATC CC-3′Nucleotides 423-436 were converted from ATGCAATTTAAACT to CGGCAATTTAACGGusing mutagenic primer 141222:

Primer 141222: (SEQ ID NO: 15)5′-GGTATTGTCCTGCAGACGGCAATTTAACGGCTTCTGCGAATCGC-3′

The resulting plasmid was designated pMT2188 (FIG. 4).

The Humicola insolens endoglucanase V coding region was transferred frompCaHj170 as a Bam HI-Sal I fragment into pMT2188 digested with Bam HIand Xho I to generate pCaHj568 (FIG. 5).

Example 4 Construction of pMJ05 Expression Vector

Expression vector pMJ05 was constructed by PCR amplifying the 915 bpHumicola insolens endoglucanase V coding region from pCaHj568 usingprimers HiEGV-F and HiEGV-R shown below.

HiEGV-F (sense): (SEQ ID NO: 16) 5′-AAGCTTAAGCATGCGTTCCTCCCCCCTCC-3′HiEGV-R (antisense): (SEQ ID NO: 17)5′-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3′

The amplification reactions (50 μl) were composed of 1× ThermoPolReaction Buffer, 0.3 mM dNTPs, 10 ng/μl pCaHj568 plasmid, 0.3 μM HiEGV-Fprimer, 0.3 μM HiEGV-R primer, and 2 U of Vent polymerase. The reactionswere incubated in an Eppendorf Mastercycler 5333 programmed as follows:5 cycles each for 30 seconds at 94° C., 30 seconds at 50° C., and 60seconds at 72° C., followed by 25 cycles each for 30 seconds at 94° C.,30 seconds at 65° C., and 120 seconds at 72° C. (5 minute finalextension). The reaction products were isolated on a 1.0% agarose gelusing TAE buffer where a 937 bp product band was excised from the geland purified using a QIAquick Gel Extraction Kit according to themanufacturer's instructions.

This 937 bp purified fragment was used as template DNA for subsequentamplifications using the following primers:

HiEGV-R (antisense): (SEQ ID NO: 18)5′-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3′ HiEGV-F-overlap (sense): (SEQ IDNO: 19) 5′-ACCGCGGACTGCGCATC ATGCGTTCCTCCCCCCTCC-3′Primer sequences in italics are homologous to 17 bp of the Trichodermareesei cbh1 promoter and underlined primer sequences are homologous to29 bp of the Humicola insolens endoglucanase V coding region. The 36 bpoverlap between the promoter and the coding sequence allowed precisefusion of the 994 bp fragment comprising the Trichoderma reesei cbh1promoter to the 918 bp fragment comprising the Humicola insolensendoglucanase V open reading frame.

The amplification reactions (50 μl) were composed of 1× ThermoPolReaction Buffer, 0.3 mM dNTPs, 1 ul of 937 bp purified PCR fragment, 0.3μM HiEGV-F-overlap primer, 0.3 μM HiEGV-R primer, and 2 U of Ventpolymerase. The reactions were incubated in an Eppendorf Mastercycler5333 programmed as follows: 5 cycles each for 30 seconds at 94° C., 30seconds at 50° C., and 60 seconds at 72° C., followed by 25 cycles eachfor 30 seconds at 94° C., 30 seconds at 65° C., and 120 seconds at 72°C. (5 minute final extension). The reaction products were isolated on a1.0% agarose gel using TAE buffer where a 945 bp product band wasexcised from the gel and purified using a QIAquick Gel Extraction Kitaccording to the manufacturer's instructions.

A separate PCR was performed to amplify the Trichoderma reesei cbh1promoter sequence extending from 994 bp upstream of the ATG start codonof the gene from Trichoderma reesei RutC30 genomic DNA using thefollowing primers (sense primer was engineered to have a Sal Irestriction site at the 5′-end):

TrCBHIpro-F (sense): (SEQ ID NO: 20) 5′-AAACGTCGACCGAATGTAGGATTGTTATC-3′TrCBHIpro-R (antisense): (SEQ ID NO: 21) 5′-GATGCGCAGTCCGCGGT-3′

The amplification reactions (50 μl) were composed of 1× ThermoPolReaction Buffer, 0.3 mM dNTPs, 100 ng of Trichoderma reesei RutC30genomic DNA, 0.3 μM TrCBHIpro-F primer, 0.3 μM TrCBHIpro-R primer, and 2U of Vent polymerase. The reactions were incubated in an EppendorfMastercycler 5333 programmed as follows: 30 cycles each for 30 secondsat 94° C., 30 seconds at 55° C., and 120 seconds at 72° C. (5 minutefinal extension). The reaction products were isolated on a 1.0% agarosegel using TAE buffer where a 998 bp product band was excised from thegel and purified using a QIAquick Gel Extraction Kit according to themanufacturer's instructions.

The 998 bp purified PCR fragment was used as template DNA for subsequentamplifications using the following primers:

TrCBHIpro-F: (SEQ ID NO: 22) 5′-AAACGTCGACCGAATGTAGGATTGTTATC-3′TrCBHIpro-R-overlap: (SEQ ID NO: 23) 5′-GGAGGGGGGAGGAACGCATGATGCGCAGTCCGCGGT-3′

Sequences in italics are homologous to 17 bp of the Trichoderma reeseicbh1 promoter and underlined sequences are homologous to 29 bp of theHumicola insolens endoglucanase V coding region. The 36 bp overlapbetween the promoter and the coding sequence allowed precise fusion ofthe 994 bp fragment comprising the Trichoderma reesei cbh1 promoter tothe 918 bp fragment comprising the Humicola insolens endoglucanase Vopen reading frame.

The amplification reactions (50 μl) were composed of 1× ThermoPolReaction Buffer, 0.3 mM dNTPs, 1 μl of 998 bp purified PCR fragment, 0.3μM TrCBH1pro-F primer, 0.3 μM TrCBH1 pro-R-overlap primer, and 2 U ofVent polymerase. The reactions were incubated in an EppendorfMastercycler 5333 programmed as follows: 5 cycles each for 30 seconds at94° C., 30 seconds at 50° C., and 60 seconds at 72° C., followed by 25cycles each for 30 seconds at 94° C., 30 seconds at 65° C., and 120seconds at 72° C. (5 minute final extension). The reaction products wereisolated on a 1.0% agarose gel using TAE buffer where a 1017 bp productband was excised from the gel and purified using a QIAquick GelExtraction Kit according to the manufacturer's instructions.

The 1017 bp Trichoderma reesei cbh1 promoter PCR fragment and the 945 bpHumicola insolens endoglucanase V PCR fragments were used as templateDNA for subsequent amplification using the following primers toprecisely fuse the 994 bp Trichoderma reesei cbh1 promoter to the 918 bpHumicola insolens endoglucanase V coding region using overlapping PCR:

TrCBHIpro-F: (SEQ ID NO: 24) 5′-AAACGTCGACCGAATGTAGGATTGTTATC-3′HiEGV-R: (SEQ ID NO: 25) 5′-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3′

The amplification reactions (50 μl) were composed of 1× ThermoPolReaction Buffer, 0.3 mM dNTPs, 0.3 μM TrCBH1pro-F primer, 0.3 μM HiEGV-Rprimer, and 2 U of Vent polymerase. The reactions were incubated in anEppendorf Mastercycler 5333 programmed as follows: 5 cycles each for 30seconds at 94° C., 30 seconds at 50° C., and 60 seconds at 72° C.,followed by 25 cycles each for 30 seconds at 94° C., 30 seconds at 65°C., and 120 seconds at 72° C. (5 minute final extension). The reactionproducts were isolated on a 1.0% agarose gel using TAE buffer where a1926 bp product band was excised from the gel and purified using aQIAquick Gel Extraction Kit according to the manufacturer'sinstructions.

The resulting 1926 bp fragment was cloned into pCR-Blunt-1′-TOPO(Invitrogen, Carlsbad, Calif.) using a ZeroBlunt TOPO PCR Cloning Kitfollowing the manufacturer's protocol. The resulting plasmid wasdigested with Not I and Sal I and the 1926 bp fragment was purified andligated into pMJ04, which was also digested with the same tworestriction enzymes, to generate pMJ05 (FIG. 6).

Example 5 Construction of pSMail30 Expression Vector

A 2586 bp DNA fragment spanning from the ATG start codon to the TAA stopcodon of the Aspergillus oryzae beta-glucosidase coding sequence (SEQ IDNO: 26 for cDNA sequence and SEQ ID NO: 27 for the deduced amino acidsequence; E. coli DSM 14240) was amplified by PCR from pJaL660 (WO2002/095014) as template with primers 993467 (sense) and 993456(antisense) shown below. A Spe I site was engineered at the 5′ end ofthe antisense primer to facilitate ligation. Primer sequences in italicsare homologous to 24 bp of the Trichoderma reesei cbh1 promoter andunderlined sequences are homologous to 22 bp of the Aspergillus oryzaebeta-glucosidase coding region.

Primer 993467: (SEQ ID NO: 28) 5′-ATAGTCAACCGCGGACTGCGCATCATGAAGCTTGGTTGGATCGAG G-3′ Primer 993456: (SEQ ID NO: 29)5′-ACTAGTTTACTGGGCCTTAGGCAGCG-3′

The amplification reactions (50 μl) were composed of Pfx AmplificationBuffer (Invitrogen, Carlsbad, Calif.), 0.25 mM dNTPs, 10 ng of pJaL660plasmid, 6.4 μM primer 993467, 3.2 μM primer 993456, 1 mM MgCl₂, and 2.5U of Pfx polymerase (Invitrogen, Carlsbad, Calif.). The reactions wereincubated in an Eppendorf Mastercycler 5333 programmed as follows: 30cycles each for 60 seconds at 94° C., 60 seconds at 55° C., and 180seconds at 72° C. (15 minute final extension). The reaction productswere isolated on a 1.0% agarose gel using TAE buffer where a 2586 bpproduct band was excised from the gel and purified using a QIAquick GelExtraction Kit according to the manufacturer's instructions.

A separate PCR was performed to amplify the Trichoderma reesei cbh1promoter sequence extending from 1000 bp upstream of the ATG start codonof the gene, using primer 993453 (sense) and primer 993463 (antisense)shown below to generate a 1000 bp PCR fragment. Primer sequences initalics are homologous to 24 bp of the Trichoderma reesei cbh1 promoterand underlined primer sequences are homologous to 22 bp of theAspergillus oryzae beta-glucosidase coding region. The 46 bp overlapbetween the promoter and the coding sequence allows precise fusion ofthe 1000 bp fragment comprising the Trichoderma reesei cbh1 promoter tothe 2586 bp fragment comprising the Aspergillus oryzae beta-glucosidaseopen reading frame.

Primer 993453: (SEQ ID NO: 30) 5′-GTCGACTCGAAGCCCGAATGTAGGAT-3′ Primer993463: (SEQ ID NO: 31) 5′-CCTCGATCCAACCAAGCTTCAT GATGCGCAGTCCGCGGTTGACTA-3′

The amplification reactions (50 μl) were composed of Pfx AmplificationBuffer, 0.25 mM dNTPs, 100 ng of Trichoderma reesei RutC30 genomic DNA,6.4 μM primer 993453, 3.2 μM primer 993463, 1 mM MgCl₂, and 2.5 U of Pfxpolymerase. The reactions were incubated in an Eppendorf Mastercycler5333 programmed as follows: 30 cycles each for 60 seconds at 94° C., 60seconds at 55° C., and 180 seconds at 72° C. (15 minute finalextension). The reaction products were isolated on a 1.0% agarose gelusing TAE buffer where a 1000 bp product band was excised from the geland purified using a QIAquick Gel Extraction Kit according to themanufacturer's instructions.

The purified fragments were used as template DNA for subsequentamplification using primer 993453 (sense) and primer 993456 (antisense)shown above to precisely fuse the 1000 bp Trichoderma reesei cbh1promoter to the 2586 bp Aspergillus oryzae beta-glucosidase fragment byoverlapping PCR.

The amplification reactions (50 μl) were composed of Pfx AmplificationBuffer, 0.25 mM dNTPs, 6.4 μM primer 99353, 3.2 μM primer 993456, 1 mMMgCl₂, and 2.5 U of Pfx polymerase. The reactions were incubated in anEppendorf Mastercycler 5333 programmed as follows: 30 cycles each for 60seconds at 94° C., 60 seconds at 60° C., and 240 seconds at 72° C. (15minute final extension).

The resulting 3586 bp fragment was digested with SalI and SpeI andligated into pMJ04, digested with the same two restriction enzymes, togenerate pSMai130 (FIG. 7).

Example 6 Construction of pSMail35

The Aspergillus oryzae beta-glucosidase coding region (WO 2002/095014,E. coli DSM 14240, minus the signal sequence, see FIG. 8, DNA sequence(SEQ ID NO: 32) and deduced amino acid sequence (SEQ ID NO: 33)) fromLys-20 to the TAA stop codon was PCR amplified from pJaL660 (WO2002/095014) as template with primer 993728 (sense) and primer 993727(antisense) shown below. Sequences in italics are homologous to 20 bp ofthe Humicola insolens endoglucanase V signal sequence and sequencesunderlined are homologous to 22 bp of the Aspergillus oryzaebeta-glucosidase coding region. A Spe I site was engineered into the 5′end of the antisense primer.

Primer 993728: (SEQ ID NO: 34) 5′-TGCCGGTGTTGGCCCTTGCCAAGGATGATCTCGCGTACTCCC-3′ Primer 993727: (SEQ ID NO: 35)5′-GACTAGTCTTACTGGGCCTTAGGCAGCG-3′

The amplification reactions (50 μl) were composed of Pfx AmplificationBuffer, 0.25 mM dNTPs, 10 ng/μl Ja1660, 6.4 μM primer 993728, 3.2 μMprimer 993727, 1 mM MgCl₂, and 2.5 U of Pfx polymerase. The reactionswere incubated in an Eppendorf Mastercycler 5333 programmed as follows:30 cycles each for 60 seconds at 94° C., 60 seconds at 55° C., and 180seconds at 72° C. (15 minute final extension). The reaction productswere isolated on a 1.0% agarose gel using TAE buffer where a 2523 bpproduct band was excised from the gel and purified using a QIAquick GelExtraction Kit according to the manufacturer's instructions.

A separate PCR amplification was performed to amplify 1000 bp of theTrichoderma reesei Cel7A cellobiohydrolase 1 promoter and 63 bp of theputative Humicola insolens endoglucanase V signal sequence (ATG startcodon to Ala-21, FIG. 9, SEQ ID NOs: 36 (DNA sequence) and 37 (deducedamino acid sequence; accession no. AAB03660 for DNA sequence), usingprimer 993724 (sense) and primer 993729 (antisense) shown below. Primersequences in italics are homologous to 20 bp of the Humicola insolensendoglucanase V signal sequence and underlined primer sequences arehomologous to 22 bp of the Aspergillus oryzae beta-glucosidase codingregion. Plasmid pMJ05, which comprises the Humicola insolensendoglucanase V coding region under the control of the cbh1 promoter,was used as a template to generate a 1063 bp fragment comprising theTrichoderma reesei cbh1 promoter/Humicola insolens endoglucanase Vsignal sequence fragment. A 42 bp of overlap was shared between theTrichoderma reesei cbh1 promoter/Humicola insolens endoglucanase Vsignal sequence and the Aspergillus oryzae coding sequence to provide aperfect linkage between the promoter and the ATG start codon of the 2523bp Aspergillus oryzae beta-glucosidase fragment.

Primer 993724: (SEQ ID NO: 38) 5′-ACGCGTCGACCGAATGTAGGATTGTTATCC-3′Primer 993729: (SEQ ID NO: 39) 5′-GGGAGTACGCGAGATCATCCTTGGCAAGGGCCAACACCGGCA-3′

The amplification reactions (50 μl) were composed of Pfx AmplificationBuffer, 0.25 mM dNTPs, 10 ng/μl pMJ05, 6.4 μM primer 993728, 3.2 μMprimer 993727, 1 mM MgCl₂, and 2.5 U of Pfx polymerase. The reactionswere incubated in an Eppendorf Mastercycler 5333 programmed as follows:30 cycles each for 60 seconds at 94° C., 60 seconds at 60° C., and 240seconds at 72° C. (15 minute final extension). The reaction productswere isolated on a 1.0% agarose gel using TAE buffer where a 1063 bpproduct band was excised from the gel and purified using a QIAquick GelExtraction Kit according to the manufacturer's instructions.

The purified overlapping fragments were used as a template foramplification using primer 993724 (sense) and primer 993727 (antisense)described above to precisely fuse the 1063 bp Trichoderma reesei cbh1promoter/Humicola insolens endoglucanase V signal sequence fragment tothe 2523 bp of Aspergillus oryzae beta-glucosidase fragment byoverlapping PCR.

The amplification reactions (50 μl) were composed of Pfx AmplificationBuffer, 0.25 mM dNTPs, 6.4 μM primer 993724, 3.2 μM primer 993727, 1 mMMgCl₂, and 2.5 U of Pfx polymerase. The reactions were incubated in anEppendorf Mastercycler 5333 programmed as follows: 30 cycles each for 60seconds at 94° C., 60 seconds at 60° C., and 240 seconds at 72° C. (15minute final extension). The reaction products were isolated on a 1.0%agarose gel using TAE buffer where a 3591 bp product band was excisedfrom the gel and purified using a QIAquick Gel Extraction Kit accordingto the manufacturer's instructions.

The resulting 3591 bp fragment was digested with Sal I and Spe I andligated into pMJ04 digested with the same restriction enzymes togenerate pSMai135 (FIG. 10).

Example 7 Expression of Aspergillus oryzae Beta-Glucosidase inTrichoderma reesei

Plasmid pSMai130, in which the Aspergillus oryzae beta-glucosidase isexpressed from the cbh1 promoter and native secretion signal (FIG. 8),or pSMai135 encoding the mature Aspergillus oryzae beta-glucosidaseenzyme linked to the Humicola insolens endoglucanase V secretion signal(FIG. 9), was introduced into Trichoderma reesei RutC30 by PEG-mediatedtransformation as described below. Both plasmids contain the Aspergillusnidulans amdS gene to enable transformants to grow on acetamide as thesole nitrogen source.

Trichoderma reesei RutC30 was cultivated at 27° C. and 90 rpm in 25 mlof YP medium (composed per liter of 10 g of yeast extract and 20 g ofBactopeptone) supplemented with 2% (w/v) glucose and 10 mM uridine for17 hours. Mycelia were collected by filtration using Millipore's VacuumDriven Disposable Filtration System (Millipore, Bedford, Mass.) andwashed twice with deionized water and twice with 1.2 M sorbitol.Protoplasts were generated by suspending the washed mycelia in 20 ml of1.2 M sorbitol containing 15 mg of Glucanex (Novozymes A/S, Bagsværd,Denmark) per ml and 0.36 units of chitinase (Sigma Chemical Co., St.Louis, Mo.) per ml and incubating for 15-25 minutes at 34° C. withgentle shaking at 90 rpm. Protoplasts were collected by centrifuging for7 minutes at 400×g and washed twice with cold 1.2 M sorbitol. Theprotoplasts were counted using a haemacytometer and re-suspended in STC(1M sorbitol, 10 mM Tris-HCl, pH 6.5, 10 mM CaCl₂) to a finalconcentration of 1×10⁸ protoplasts per ml. Excess protoplasts werestored in a Cryo 1° C. Freezing Container (Nalgene, Rochester, N.Y.) at−80° C.

Approximately 7 μg of Pme I digested expression plasmid (pSMai130 orpSMai135) was added to 100 μl of protoplast solution and mixed gently,followed by 260 μl of PEG buffer (60% PEG-4000, 10 mM Tris-HCl, pH 6.5,10 mM CaCl₂), mixed, and incubated at room temperature for 30 minutes.STC (3 ml) was then added and mixed and then the transformation solutionwas plated onto COVE plates (composed per liter of 342.3 g of sucrose,10 ml of 1 M acetamide solution, 10 ml of 1.5 M CsCl solution, 25 g ofagar, and 20 ml of Cove salts solution; Cove salts solution was composedper liter of 26 g of KCl, 26 g of MgSO₄.7H₂O, 76 g of KH₂PO₄, and 50 mlof Cove trace metals solution; Cove trace metals solution was composedper liter of 0.04 g of Na₂B₄O₇.10H₂O, 0.4 g of CuSO₄.5H₂O, 1.2 g ofFeSO₄.7H₂O, 0.7 g of MnSO₄.H₂O, 0.8 g of Na₇MoO₇.2H₂O, and 10 g ofZnSO₄.7H₂O). The plates were incubated at 28° C. for 5-7 days.Transformants were subcultured onto COVE2 plates (composed per liter of30 g of sucrose, 10 ml of 1 M acetamide solution, 20 ml of Cove saltssolution, and 25 g of agar) and grown at 28° C.

One hundred and ten amdS positive transformants were obtained withpSMai130 and 65 transformants with pSMai135. Twenty pSMai130 (nativesecretion signal) and 67 pSMai135 (heterologous secretion signal)transformants were subcultured onto fresh plates containing acetamideand allowed to sporulate for 7 days at 28° C.

The 20 pSMA130 and 67 pSMA135 Trichoderma reesei transformants werecultivated in 125 ml baffled shake flasks containing 25 ml ofcellulase-inducing medium at pH 6.0 inoculated with spores of thetransformants and incubated at 28° C. and 200 rpm for 7 days.Trichoderma reesei RutC30 was run as a control. Culture broth sampleswere removed at day 7. One ml of each culture broth was centrifuged at15,700×g for 5 minutes in a micro-centrifuge and the supernatantstransferred to new tubes. Samples were stored at 4° C. until enzymeassay. The supernatants were assayed for beta-glucosidase activity usingp-nitrophenyl-beta-D-glucopyranoside as substrate, as described below.

Beta-glucosidase activity was determined at ambient temperature using 25μl aliquots of culture supernatants, diluted 1:10 in 50 mM succinate pH5.0, using 200 μl of 0.5 mg/ml p-nitrophenyl-beta-D-glucopyranoside assubstrate in 50 mM succinate pH 5.0. After 15 minutes incubation thereaction was stopped by adding 100 μl of 1 M Tris-HCl pH 8.0 and theabsorbance was read spectrophotometrically at 405 nm.

One unit of beta-glucosidase activity corresponded to production of 1μmol of p-nitrophenyl per minute per liter at pH 5.0, ambienttemperature. Aspergillus niger beta-glucosidase (Novozyme 188, NovozymesA/S, Bagsværd, Denmark) was used as an enzyme standard.

All 20 SMA130 transformants exhibited equivalent beta-glucosidaseactivity to that of the host strain, Trichoderma reesei RutC30. Incontrast, a number of SMA135 transformants showed beta-glucosidaseactivities several fold more than that of Trichoderma reesei RutC30.Transformant SMA135-04 produced the highest beta-glucosidase activity,having seven times greater beta-glucosidase activity than produced byTrichoderma reesei RutC30 as a control.

SDS polyacrylamide electrophoresis was carried out using CriterionTris-HCl (5% resolving) gels (BioRad, Hercules, Calif.) with TheCriterion System (BioRad, Hercules, Calif.). Five μl of day 7supernatants (see above) were suspended in 2× concentration of LaemmliSample Buffer (BioRad, Hercules, Calif.) and boiled for 3 minutes in thepresence of 5% beta-mercaptoethanol. The supernatant samples were loadedonto a polyacrylamide gel and subjected to electrophoresis with 1×Tris/Glycine/SDS as running buffer (BioRad, Hercules, Calif.). Theresulting gel was stained with BioRad's Bio-Safe Coomassie Stain.

No beta-glucosidase protein was visible by SDS-PAGE for the Trichodermareesei SMA130 transformant culture broth supernatants. In contrast, 26of the 38 Trichoderma reesei SMA135 transformants produced a protein ofapproximately 110 kDa that was not visible in Trichoderma reesei RutC30as control. Transformant Trichoderma reesei SMA135-04 produced thehighest level of beta-glucosidase.

Example 8 Fermentation of Trichoderma reesei SMA135-04

Fermentations of Trichoderma reesei SMA135-04 were performed todetermine the production level of beta-glucosidase activity. Trichodermareesei RutC30 (host strain) was run as a control. Spores of Trichodermareesei SMA135-04 were inoculated into 500 ml shake flasks, containing100 ml of inoculum medium composed per liter of 20 g of glucose, 10 g ofcorn steep solids, 1.45 g of (NH₄)₂SO₄, 2.08 g of KH₂PO₄, 0.36 g ofCaCl₂.2H₂O, 0.42 g of MgSO₄.7H₂O, and 0.2 ml of trace metals solution.The trace metals solution was composed per liter of 216 g of FeCl₃.6H₂O,58 g of ZnSO₄.7H₂O, 27 g of MnSO₄.H₂O, 10 g of CuSO₄.5H₂O, 2.4 g ofH₃BO₃, and 336 g of citric acid. The flasks were placed into an orbitalshaker at 28° C. for approximately 48 hours at which time 50 ml of theculture was inoculated into 1.8 liters of fermentation medium composedper liter of 4 g of glucose, 10 g of corn steep solids, 30 g ofcellulose, 2.64 g of CaCl₂.2H₂O, 3.8 g of (NH₄)₂SO₄, 2.8 g of KH₂PO₄,1.63 g of MgSO₄.7H₂O, 0.75 ml of trace metals solution (described above)in a 2 liter fermentation vessel. The fermentations were run at a pH of5.0, 28° C., with minimum dissolved oxygen at a 25% at a 1.0 VVM airflow and an agitation of 1100. Feed medium was delivered into thefermentation vessel at 18 hours with a feed rate of 3.6 g/hour for 33hours and then 7.2 g/hour. The fermentations ran for 165 hours at whichtime the final fermentation broths were centrifuged and the supernatantsstored at −20° C. until beta-glucosidase activity assay using theprocedure described in Example 7.

Beta-glucosidase activity on the Trichoderma reesei SMA135-04fermentation sample was determined to be approximately eight timesgreater than that produced by Trichoderma reesei RutC30.

Example 9 PCS Hydrolysis Using Fresh Fermentation Samples

PCS hydrolysis reactions were formulated using washed and milled cornstover that was pretreated with dilute sulfuric acid at elevatedtemperature and pressure. The following conditions were used for thepretreatment: acid concentration—1.4 wt %; temperature—165° C.; pressure107 psi; time—8 minutes. Prior to enzymatic hydrolysis, the pretreatedcorn stover (PCS) was washed with a large volume of distilled-deionized(DDI) water on a glass filter. The dry weight of the water-washed PCSwas found to be 24.54%. The water-insoluble solids in PCS contained56.5% cellulose, 4.6% hemicellulose, and 28.4% lignin.

Prior to enzymatic hydrolysis, a suspension of milled PCS in DDI waterwas prepared as follows: DDI water-washed PCS was additionally washedwith 95% ethanol on a 22 μm Millipore Filter (6P Express Membrane,Stericup), and then milled using a coffee-grinder to reduce the particlesize. Dry weight of the milled PCS was found to be 41.8%. Milled PCS waswashed with DDI water three times in order to remove the ethanol. Aftereach washing, the suspension was centrifuged at 17,000×g for 10 minutesat 4° C. to separate the solids. Finally, DDI water was added to themilled water-washed solids to make 20 mg/ml suspension. The suspensionwas stored at 4° C. and used for 1 ml scale PCS hydrolysis at finalconcentration of 10 mg/ml.

Trichoderma reesei strains were grown in two-liter Applikon laboratoryfermentors using a cellulase producing medium at 28° C., pH 4.5, and agrowth time of approximately 120 hours. The cellulose producing mediumwas composed per liter of 5 g of glucose, 10 g of corn steep solids,2.08 g of CaCl₂, 3.87 g of (NH₄)₂SO₄, 2.8 g of KH₂PO₄, 1.63 g ofMgSO₄.7H₂O, 0.75 ml of trace metals solution, and 1.8 ml of pluronicwith a feed of 20 g of cellulose per liter. The trace metals solutionwas composed per liter of 216 g of FeCl₃.6H₂O, 58 g of ZnSO₄.7H₂O, 27 gof MnSO₄.H₂O, 10 g of CuSO₄.5H₂O, 2.4 g of H₃BO₃, and 336 g of citricacid.

The procedure for preparation of whole fermentation broth (WB) andcell-free broth (CB) samples is outlined as follows. Briefly, two 50 mlaliquots of Trichoderma reesei culture were harvested aseptically inconical centrifuge tubes. One of these tubes was designated as the WBenzyme preparation without further treatment, and the other wascentrifuged twice to remove cells and insoluble material to yield a CBenzyme sample. The first centrifugation was at low speed (1800×g for 10minutes), and the second was at higher speed (12,000×g for 15 minutes).

PCS (10 mg/ml, 56.5% cellulose) was enzymatically hydrolyzed at 50° C.in 0.05 M sodium acetate buffer (pH 5.0) with intermittent mixing. Twotypes of enzyme preparations were used in these experiments: (a) Wholefermentation broth (WB) and (b) centrifuged fermentation broth (CB) asdefined above. In one series of experiments CB that was centrifugedprior to storage (CB-A) for two weeks at 4° C. was compared with broththat was centrifuged after storage (CB-B). Four enzyme doses weretested: 2.5, 5.0, 10, and 20 mg/g of PCS. These doses were based onestimated protein concentrations of 60 g/L for standard lab-scalefermentations. The volume of each reaction was 1 ml in MicroWell96™ deepwell plates (Fisher Scientific, Pittsburg, Pa.). At specified timepoints (1, 3, 6, 9, 12, 24, 48, 72, 96, and 120 hours) 20 μl aliquotswere removed from the microplates using an 8-channel pipettor, and addedto 180 μl of alkaline mixture (0.102 M Na₂CO₃+0.058 M NaHCO₃) in a96-well flat-bottomed plate (Millipore, Billerica, Mass.) to terminatethe reaction. The samples were centrifuged at 1800×g for 15 minutes toremove unreacted PCS residue. After appropriate dilutions, the filtrateswere analyzed for reducing sugars (RS) using a microplate assay (seebelow).

The concentrations of reducing sugars (RS) in hydrolyzed PCS sampleswere measured using a p-hydroxybenzoic acid hydrazide (PHBAH) assay(Lever, 1972, Anal. Biochem, 47: 273-279), which was modified andadapted to a 96-well microplate format. Before the assay, the analyzedsamples were diluted in water to bring the RS concentration into the0.005-0.200 mg/ml range.

A 90 μl aliquot of each diluted reaction sample was placed in a 96-wellconical-bottomed microplate (Corning Inc., Costar, clear polycarbonate).The reactions were started by addition of 60 μl of 1.25% PHBAH in 2%sodium hydroxide. Each assay plate was heated on a custom-made heatingblock for 10 minutes at 95° C., and allowed to cool at room temperature.After cooling, 60 μl of water was added to each well. A 100 μl aliquotwas removed and transferred to a flat-bottomed 96-well plate (CorningInc., Costar, medium binding polystyrene), and the absorbance at 405 nm(A₄₀₅) was measured using an UltraMark Microplate Reader (Bio-Rad,Hercules, Calif.). The A₄₀₅ values were translated into glucoseequivalents using a standard curve. In order to increase the statisticalprecision of the assays, 32 replicates were done for each time point ateach enzyme dose.

Standard curves were generated with eight glucose standards (0.000,0.005, 0.010, 0.020, 0.030, 0.050, 0.075, and 0.100 mg/ml), which weretreated similarly to the samples. Glucose standards were prepared bydiluting a 10 mg/ml stock glucose solution with sodiumcarbonate/bicarbonate mixture (0.102 M Na₂CO₃+0.058 M NaHCO₃). Eightreplicates of each standard were done to increase precision of theassays. The average correlation coefficient for the standard curves wasgreater than 0.99.

Glucose concentrations in each hydrolyzed sample were measured using anenzyme-linked assay method in which 50 μl of each diluted PCShydrolysate were mixed with 100 μl of assay buffer (100 mM MOPS, pH 7,0.01% Tween-20) and 150 μl of glucose assay reagent. The assay reagentcontained the following ingredients (per liter): 0.5511 g of ATP, 0.9951g of NAD, 0.5176 g of MgSO₄.7H₂O, 1000 Units/L hexokinase Type 300(Sigma Chemical Co., St. Louis, Mo.), 1000 Units/L ofglucose-6-phosphate dehydrogenase (Sigma Chemical Co., St. Louis, Mo.),0.1 g of Tween-20, and 20.9 g of MOPS, pH 7.0. The reactions wereincubated for 30 minutes at ambient temperature, and the absorbance wasmeasured at 340 nm. Background absorbance was subtracted based on a zeroglucose control, and the glucose concentrations were determined withrespect to a standard curve generated with glucose concentrationsranging from 0.00 to 0.25 mg/ml.

The mean RS yield was calculated using data from all replicates at aparticular enzyme dose and incubation time. Standard error of the mean(SEM) was calculated as the standard deviation divided by thesquare-root n, the number of replicates. The degree of celluloseconversion to reducing sugar (RS yield, percent) was calculated usingthe following equation:

$\begin{matrix}{{{RS}\mspace{14mu} {Yield}_{(\%)}} = {{RS}_{({{mg}/{ml}})} \times 100 \times {162/\left( {5.65_{({{mg}/{ml}})} \times 180} \right)}}} \\{= {{RS}_{({{mg}/{ml}})} \times {100/\left( {5.65_{({{mg}/{ml}})} \times 1.111} \right)}}}\end{matrix}$

In this equation, RS is the concentration of reducing sugar in solutionmeasured in glucose equivalents (mg/ml), 5.65 mg/ml is the initialconcentration of cellulose, and the factor 1.111 reflects the weightgain in converting cellulose to glucose.

The probability that WB and CB data points represented statisticallydifferent populations was estimated using a Student t-test (with unequalvariance) at each time point.

As shown in FIG. 11, the use of freshly harvested enzyme samples (WB andCB) produced PCS hydrolysis profiles that were nearly identical. Theseprofiles could not be differentiated with a Student t-test suggestingthat they were statistically indistinguishable. Using enzyme samplesfrom Trichoderma reesei RutC30, the final RS yields ranged fromapproximately 30% conversion of the total glucan at the lowest enzymedose to about 50% at the highest dose (FIG. 11). When the concentrationof glucose was measured instead of reducing sugars, a similar pictureemerged in that the glucose yields were comparable regardless of whetherWB or CB was used (FIG. 12). However, it may be noteworthy that theglucose yields were approximately 20 to 25% lower than the reducingsugar concentrations suggesting that beta-glucosidase might be alimiting enzyme activity under these conditions.

In an effort to convert a higher percentage of RS to glucose, enzymesamples were deployed from the recombinant Trichoderma reesei strainSMA135-04 which expresses an Aspergillus oryzae beta-glucosidase gene.When these preparations with elevated β-glucosidase activity wereemployed, several differences were observed based on a comparison to theresults from Trichoderma reesei RutC30 enzyme samples. First, within thelimits of systematic and experimental errors the PCS hydrolysis curvesfor WB and CB were very similar (FIG. 13). Second, at the lowest enzymedose (2.5 mg/g of PCS) the final RS yields obtained from Trichodermareesei SAM135-04 enzyme samples were approximately 40% of the totalglucan hydrolyzed compared to 30% for Trichoderma reesei RutC30 enzyme.Third, the final RS titers at higher enzyme doses were essentiallyunchanged compared to those obtained when using WB and CB preparationsderived from Trichoderma reesei RutC30 (FIG. 13). The reasons for thisphenomenon are unclear, but it may reflect either thermal inactivationof endoglucanases and cellobiohydrolases during prolonged incubation at50° C. or end product inhibition of the Aspergillus oryzaeβ-glucosidase. On the basis of these comparisons the data consistentlysuggested that there is little difference between WB and CB hydrolysisprofiles. Both the reaction kinetics and final RS titers appeared to besimilar.

When the RS and glucose yields generated from WB and CB samples ofTrichoderma reesei SMA135-04 were compared to those obtained fromTrichoderma reesei RutC30 preparations, we observed that a higherpercentage of RS was converted to glucose by SMA135-04 enzyme samples(FIG. 14). This was not unexpected since Trichoderma reesei SMA135-04produces higher levels of β-glucosidase than Trichoderma reesei RutC30.Interestingly, at early time points (up to 24 hours), the RS and glucoselevels generated from the Trichoderma reesei SMA135-04 enzymepreparations differed by only a few percent (FIG. 14). However, duringlater stages of the reactions, the RS and glucose curves divergeperceptibly, suggesting that the beta-glucosidase activity may bedeclining in the later stages of PCS hydrolysis under these conditions.This was particularly apparent at later times for the highest enzymedose (20 mg/g of PCS). In addition, it appeared that WB wasoutperforming CB in generation of both RS and glucose over this sametime period. A Student t-test predicted that the variance in RS valueswas statistically significant (P<0.05) over the time frame of 48-120hours. Whether the observed difference in performance can be attributedto specific enzyme(s) or non-specific effects attributed to the presenceof the mycelia is unknown. However, it should be noted that thephenomenon was not observed when using WB and CB enzyme samples fromTrichoderma reesei RutC30 (FIG. 15), suggesting instability of theheterologous Aspergillus oryzae beta-glucosidase expressed byTrichoderma reesei SMA135-04 during prolonged incubation.

Example 10 PCS Hydrolysis Using Fermentation Broth Stored for Two Weeksat 4° C.

A biomass-to-ethanol process scheme involving on-site enzymemanufacturing should incorporate enough flexibility to allow for finitestorage of enzyme preparations without significant loss of potency.Therefore, whether prolonged cold storage of enzyme samples affectedtheir performance in microtiter-scale PCS hydrolysis reactions wasinvestigated. In addition to WB, two types of CB preparations weretested. CB-A samples were centrifuged at time of harvest and stored at4° C. as cell-free supernatant; CB-B preparations were stored at 4° C.as whole broth, then centrifuged to remove cells at the time of theassay.

The PCS hydrolysis reactions were performed as described in Example 9.

FIGS. 16 and 17 shows that the hydrolysis curves for WB, CB-A, and CB-Bwere principally similar. Statistical analyses using Student t-testssupported that these data points were not appreciably different.Furthermore, the final RS yields obtained using enzyme samples that werestored for two weeks were essentially the same as those obtained fromthe use of fresh fermentation broth. As was observed with fresh brothmaterial, the lowest dose of Trichoderma reesei SMA135-04 enzyme (2.5mg/g of PCS) gave slightly higher RS yields than the same dose of Tv10material, ostensibly because of higher beta-glucosidase levels producedby Trichoderma reesei S A135-04.

These results can be summarized as follows:

1. WB appears to perform as well as CB for hydrolysis of PCS under theassay conditions described in this series of experiments.

2. It is possible to store WB and CB enzyme samples from Trichodermareesei fermentations for at least two weeks at 4° C. without significantloss of potency in our hydrolysis assay.

3. CB may be processed from WB that has been stored for two weeks at 4°C. without appreciable loss of activity in our hydrolysis assay.

Collectively, the results suggested that it is possible to achievesimilar PCS hydrolysis results using WB instead of fractionated orformulated culture filtrates. It should be highlighted that thehydrolysis experiments were dosed on an equal volume basis, and theywere not normalized on the basis of enzyme activity or proteinconcentration. Consequently, it was surprising to have observedequivalent performance of WB and CB dosed in this manner, because thefungal cell mass accounted for approximately 20-30% of the volume in WB.This implied that the effective dose of extracellular enzyme in the WBpreparations was about 20-30% lower than that of CB.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

What is claimed is:
 1. A method for degrading or converting plant cellwall polysaccharides into one or more products, comprising: treating theplant cell wall polysaccharides with an effective amount of a spentwhole fermentation broth of a recombinant microorganism, wherein therecombinant microorganism expresses one or more heterologous genesencoding enzymes which degrade or convert the plant cell wallpolysaccharides into the one or more products.
 2. The method of claim 1,wherein the one or more heterologous genes encode enzymes selected fromthe group consisting of a cellulase, endoglucanase, cellobiohydrolase,and beta-glucosidase.
 3. The method of claim 1, wherein the heterologousgene encodes a cellulase.
 4. The method of claim 1, wherein theheterologous gene encodes an endoglucanase.
 5. The method of claim 1,wherein the heterologous gene encodes a cellobiohydrolase.
 6. The methodof claim 1, wherein the heterologous gene encodes a beta-glucosidase. 7.The method of claim 1, wherein the one or more heterologous genes encodeenzymes further selected from the group consisting of a glucohydrolase,xyloglucanase, xylanase, xylosidase, alpha-arabinofuranosidase,alpha-glucuronidase, acetyl xylan esterase, mannanase, mannosidase,alpha-galactosidase, mannan acetyl esterase, galactanase, arabinanase,pectate lyase, pectin lyase, pectate lyase, polygalacturonase, pectinacetyl esterase, pectin methyl esterase, alpha-arabinofuranosidase,beta-galactosidase, galactanase, arabinanase, alpha-arabinofuranosidase,rhamnogalacturonase, rhamnogalacturonan lyase, rhamnogalacturonan acetylesterase, xylogalacturonosidase, xylogalacturonase, rhamnogalacturonanlyase, lignin peroxidase, manganese-dependent peroxidase, hybridperoxidase, and laccase.
 8. The method of claim 1, wherein the spentwhole fermentation broth of the recombinant microorganism issupplemented by the addition of one or more enzymes selected from thegroup consisting of a cellulase, endoglucanase, cellobiohydrolase, andbeta-glucosidase.
 9. The method of claim 1, further comprisingrecovering the one or more products obtained from the degraded orconverted plant cell wall polysaccharides.
 10. The method of claim 9,wherein the product is a sugar.
 11. A method for producing one or moresubstances, comprising: (a) saccharifying plant cell wallpolysaccharides with an effective amount of a spent whole fermentationbroth of a recombinant microorganism, wherein the recombinantmicroorganism expresses one or more heterologous genes encoding enzymeswhich degrade or convert the plant cell wall polysaccharides intosaccharified material; (b) fermenting the saccharified material of step(a) with one or more fermenting microoganisms; and (c) recovering theone or more organic substances from the fermentation.
 12. The method ofclaim 11, wherein the one or more heterologous genes encode enzymesselected from the group consisting of a cellulase, endoglucanase,cellobiohydrolase, and beta-glucosidase.
 13. The method of claim 11,wherein the heterologous gene encodes a cellulase.
 14. The method ofclaim 11, wherein the heterologous gene encodes an endoglucanase. 15.The method of claim 11, wherein the heterologous gene encodes acellobiohydrolase.
 16. The method of claim 11, wherein the heterologousgene encodes a beta-glucosidase.
 17. The method of claim 11, wherein theone or more heterologous genes encode enzymes further selected from thegroup consisting of a glucohydrolase, xyloglucanase, xylanase,xylosidase, alpha-arabinofuranosidase, alpha-glucuronidase, acetyl xylanesterase, mannanase, mannosidase, alpha-galactosidase, mannan acetylesterase, galactanase, arabinanase, pectate lyase, pectin lyase, pectatelyase, polygalacturonase, pectin acetyl esterase, pectin methylesterase, alpha-arabinofuranosidase, beta-galactosidase, galactanase,arabinanase, alpha-arabinofuranosidase, rhamnogalacturonase,rhamnogalacturonan lyase, rhamnogalacturonan acetyl esterase,xylogalacturonosidase, xylogalacturonase, rhamnogalacturonan lyase,lignin peroxidase, manganese-dependent peroxidase, hybrid peroxidase,and laccase.
 18. The method of claim 11, wherein the spent wholefermentation broth of the recombinant microorganism is supplemented bythe addition of one or more enzymes selected from the group consistingof a cellulase, endoglucanase, cellobiohydrolase, and beta-glucosidase.19. The method of claim 11, wherein steps (a) and (b) are performedsimultaneously in a simultaneous saccharification and fermentation. 20.The method of claim 11, wherein the one or more substances are selectedfrom the group consisting of an alcohol, an organic acid, a ketone, analdehyde, an amino acid, a gas, and a combination thereof.